Methods for treatment of cardiovascular disease

ABSTRACT

This invention pertains to methods and compositions for the diagnosis and treatment of cardiovascular conditions. More specifically, the invention relates to isolated molecules that can be used to diagnose and/or treat cardiovascular conditions including cardiac hypertrophy, myocardial infarction, stroke, arteriosclerosis, and heart failure.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/167,143, filed on Jul. 2, 2008, which is a continuation of U.S.patent application Ser. No. 10/024,607 filed on Nov. 8, 2001, issued asU.S. Pat. No. 7,432,060, which claims priority under 35 U.S.C. §119(e)from Provisional U.S. Patent Application Ser. No. 60/247,457 filed onNov. 9, 2000. The entire contents of each of which are hereinincorporated by reference.

GOVERNMENT SUPPORT

This invention was made with government support under grant numberHL054759 awarded by The National Institute of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

This invention relates to methods and compositions for the diagnosis andtreatment of cardiovascular conditions. More specifically, the inventionrelates to isolated molecules that can be used to treat cardiovascularconditions including cardiac hypertrophy, myocardial infarction, stroke,arteriosclerosis, and heart failure.

BACKGROUND OF THE INVENTION

Despite significant advances in therapy, cardiovascular disease remainsthe single most common cause of morbidity and mortality in the developedworld. Thus, prevention and therapy of cardiovascular conditions such asmyocardial infarction and stroke is an area of major public healthimportance. Currently, several risk factors for future cardiovasculardisorders have been described and are in wide clinical use in thedetection of individuals at high risk. Such screening tests includeevaluations of total and HDL cholesterol levels. However, a large numberof cardiovascular disorders occur in individuals with apparently low tomoderate risk profiles, and ability to identify such patients islimited. Moreover, accumulating data suggests that the beneficialeffects of certain preventive and therapeutic treatments for patients atrisk for or known to have cardiovascular disorders differs in magnitudeamong different patient groups. At this time, however, data describingdiagnostic tests to determine whether certain therapies can be expectedto be more or less effective are lacking.

SUMMARY OF THE INVENTION

This invention provides methods and compositions for the diagnosis andtreatment of cardiovascular conditions. More specifically, a number ofgenes were identified that are upregulated in cardiac cells when thecells are subjected to mechanically-induced deformation. In view ofthese discoveries, it is believed that the molecules of the presentinvention can be used to treat vascular and cardiovascular conditionsincluding cardiac hypertrophy, myocardial infarction, stroke,arteriosclerosis, and heart failure.

Additionally, methods for using these molecules in the diagnosis of anyof the foregoing vascular and cardiovascular conditions, are alsoprovided.

Furthermore, compositions useful in the preparation of therapeuticpreparations for the treatment of the foregoing conditions, are alsoprovided.

The present invention thus involves, in several aspects, polypeptides,isolated nucleic acids encoding those polypeptides, functionalmodifications and variants of the foregoing, useful fragments of theforegoing, as well as therapeutics and diagnostics relating thereto.

According to one aspect of the invention, a method of diagnosing acondition characterized by aberrant expression of a nucleic acidmolecule or an expression product thereof (or of unique fragments of theforegoing molecules thereof), is provided. The method involvescontacting a biological sample from a subject with an agent, whereinsaid agent specifically binds to said nucleic acid molecule, anexpression product thereof, or a fragment of an expression productthereof, and measuring the amount of bound agent and determiningtherefrom if the expression of said nucleic acid molecule or of anexpression product thereof is aberrant, aberrant expression beingdiagnostic of the disorder, wherein the nucleic acid molecule is atleast one nucleic acid molecule selected from the group consisting ofFit-1 (SEQ ID NOs: 1 and 2 for Fit-1S; SEQ ID NOs: 3 and 4 for Fit-1M),vacuolar ATPase (SEQ ID NOs: 5 and 6), CD44 (SEQ ID NOs: 7 and 8), Lot-1(SEQ ID NOs: 9 and 10), AA892598 (SEQ ID NO: 11), and Mrg-1 (SEQ ID NO:12). In some embodiments, the disorder is a cardiovascular conditionselected from the group consisting of myocardial infarction, stroke,arteriosclerosis, and heart failure. In one embodiment, the disorder iscardiac hypertrophy. In certain embodiments, biological samples includebiopsy samples, and biological fluids such as blood.

According to still another aspect of the invention, a method fordetermining a stage (e.g., regression, progression or onset) of acardiovascular condition in a subject characterized by aberrantexpression of a nucleic acid molecule or an expression product thereof(or of unique fragments of the foregoing molecules thereof), isprovided. The method involves monitoring a sample from a patient for aparameter selected from the group consisting of (i) a nucleic acidmolecule selected from the group consisting of Fit-1, vacuolar ATPase,CD44, Lot-1, AA892598, and Mrg-1 (or a unique fragment thereof), (ii) apolypeptide encoded by the nucleic acid, (iii) a peptide derived fromthe polypeptide (or of a unique fragment thereof), and (iv) an antibodywhich selectively binds the polypeptide or peptide (or a unique fragmentthereof), as a determination of a stage (e.g., regression, progressionor onset) of said cardiovascular condition in the subject. In someembodiments, the sample is a biological fluid or a tissue as describedin any of the foregoing embodiments. In certain embodiments, the step ofmonitoring comprises contacting the sample with a detectable agentselected from the group consisting of (a) an isolated nucleic acidmolecule which selectively hybridizes is under stringent conditions tothe nucleic acid molecule of (i), (b) an antibody which selectivelybinds the polypeptide of (ii), or the peptide of (iii), and (c) apolypeptide or peptide which binds the antibody of (iv). The antibody,polypeptide, peptide, or nucleic acid can be labeled with a radioactivelabel or an enzyme. In further embodiments, the method further comprisesassaying the sample for the peptide. In still further embodiments,monitoring the sample occurs over a period of time.

According to another aspect of the invention, a kit is provided. The kitcomprises a package containing an agent that selectively binds to any ofthe foregoing isolated nucleic acids (Fit-1, vacuolar ATPase, CD44,Lot-1, AA892598, and Mrg-1), or expression products thereof, and acontrol for comparing to a measured value of binding of said agent anyof the foregoing isolated nucleic acids or expression products thereof.In some embodiments, the control is a predetermined value for comparingto the measured value. In certain embodiments, the control comprises anepitope of the expression product of any of the foregoing isolatednucleic acids.

According to one aspect of the invention, a method for treating acardiovascular condition, is provided. The method involves administeringto a subject in need of such treatment a molecule selected from thegroup consisting of Fit-1 (alternatively referred to herein as T1/ST2),CD44, Lot-1, AA892598, and Mrg-1, in an amount effective to treat thecardiovascular condition. In certain embodiments, the cardiovascularcondition is selected from the group consisting of myocardialinfarction, stroke, arteriosclerosis, and heart failure. In oneembodiment, the molecule administered is vacuolar ATPase. In someembodiments, the method further comprises co-administering an agentselected from the group consisting of an anti-inflammatory agent, ananti-thrombotic agent, an anti-platelet agent, a fibrinolytic agent, alipid reducing agent, a direct thrombin inhibitor, a glycoproteinIIb/IIIa receptor inhibitor, an agent that binds to cellular adhesionmolecules and inhibits the ability of white blood cells to attach tosuch molecules, a calcium channel blocker, a beta-adrenergic receptorblocker, a cyclooxygenase-2 inhibitor, or an angiotensin systeminhibitor.

According to another aspect of the invention, a method for treatingcardiac hypertrophy, is provided. The method involves administering to asubject in need of such treatment an agent that increases expression ofa nucleic acid molecule selected from the group consisting of Fit-1,vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1, or an expressionproduct thereof, in an amount effective to treat cardiac hypertrophy inthe subject.

According to a further aspect of the invention, a method for treating asubject to is reduce the risk of a cardiovascular condition developingin the subject, is provided. The method involves administering to asubject that expresses decreased levels of a molecule selected from thegroup consisting of Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, andMrg-1, an agent for reducing the risk of the cardiovascular disorder inan amount effective to lower the risk of the subject developing a futurecardiovascular disorder, wherein the agent is an anti-inflammatoryagent, an anti-thrombotic agent, an anti-platelet agent, a fibrinolyticagent, a lipid reducing agent, a direct thrombin inhibitor, aglycoprotein IIb/IIIa receptor inhibitor, an agent that binds tocellular adhesion molecules and inhibits the ability of white bloodcells to attach to such molecules, a calcium channel blocker, abeta-adrenergic receptor blocker, a cyclooxygenase-2 inhibitor, or anangiotensin system inhibitor, or an agent that increases expression of amolecule selected from the group consisting of Fit-1, vacuolar ATPase,CD44, Lot-1, AA892598, and Mrg-1.

According to one aspect of the invention, a method for identifying acandidate agent useful in the treatment of a cardiovascular condition,is provided. The method involves determining expression of a set ofnucleic acid molecules in a cardiac cell or tissue under conditionswhich, in the absence of a candidate agent, permit a first amount ofexpression of the set of nucleic acid molecules, wherein the set ofnucleic acid molecules comprises at least one nucleic acid moleculeselected from the group consisting of Fit-1, vacuolar ATPase, CD44,Lot-1, AA892598, and Mrg-1, contacting the cardiac cell or tissue withthe candidate agent, and detecting a test amount of expression of theset of nucleic acid molecules, wherein an increase in the test amount ofexpression in the presence of the candidate agent relative to the firstamount of expression indicates that the candidate agent is useful in thetreatment of the cardiovascular condition. In certain embodiments, thecardiovascular condition is selected from the group consisting ofcardiac hypertrophy (e.g., maladaptive hypertrophy), myocardialinfarction, stroke, arteriosclerosis, and heart failure. In someembodiments, the set of nucleic acid molecules comprises at least two,at least three, at least four, or even at least five nucleic acidmolecules, each selected from the group consisting of Fit-1, vacuolarATPase, CD44, Lot-1, AA892598, and Mrg-1.

According to another aspect of the invention, a pharmaceuticalcomposition is provided. The composition comprises an agent comprisingan isolated nucleic acid molecule selected from the group consisting ofFit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1, or anexpression product thereof, in a pharmaceutically effective amount totreat a cardiovascular condition, and a pharmaceutically acceptablecarrier. In some embodiments, is the agent is an expression product ofthe isolated nucleic acid molecule selected from the group consisting ofFit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1. In certainembodiments, the cardiovascular condition is selected from the groupconsisting of cardiac hypertrophy, myocardial infarction, stroke,arteriosclerosis, and heart failure.

According to a further aspect of the invention, methods for preparingmedicaments useful in the treatment of a cardiovascular condition arealso provided.

According to still another aspect of the invention, a solid-phasenucleic acid molecule array, is provided. The array consists essentiallyof a set of nucleic acid molecules, expression products thereof, orfragments (of either the nucleic acid or the polypeptide molecule)thereof, wherein at least two and as many as all of the nucleic acidmolecules selected from the group consisting of Fit-1, vacuolar ATPase,CD44, Lot-1, AA892598, and Mrg-1 (including expression products thereof,or fragments thereof), are fixed to a solid substrate. In someembodiments, the solid-phase array further comprises at least onecontrol nucleic acid molecule. In certain embodiments, the set ofnucleic acid molecules comprises at least three, at least four, or evenat least five nucleic acid molecules, each selected from the groupconsisting of Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1.In preferred embodiments, the set of nucleic acid molecules comprises amaximum number of 100 different nucleic acid molecules. In importantembodiments, the set of nucleic acid molecules comprises a maximumnumber of 10 different nucleic acid molecules.

In certain embodiments, the solid substrate includes a material selectedfrom the group consisting of glass, silica, aluminosilicates,borosilicates, metal oxides such as alumina and nickel oxide, variousclays, nitrocellulose, and nylon. Preferably the substrate is glass. Insome embodiments, the nucleic acid molecules are fixed to the solidsubstrate by covalent bonding.

These and other objects of the invention will be described in furtherdetail in connection with the detailed description of the invention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the nucleotide sequence of the rat Fit-1S cDNA.

SEQ ID NO:2 is the predicted amino acid sequence of the translationproduct of rat Fit-1S cDNA (SEQ ID NO:1).

SEQ ID NO:3 is the nucleotide sequence of the rat Fit-1M cDNA.

SEQ ID NO:4 is the predicted amino acid sequence of the translationproduct of the rat Fit-1M cDNA (SEQ ID NO:3).

SEQ ID NO:5 is the nucleotide sequence of the rat vacuolar ATPase cDNA(GenBank Acc. No. Y12635).

SEQ ID NO:6 is the predicted amino acid sequence of the translationproduct of the rat vacuolar ATPase cDNA (SEQ ID NO:5).

SEQ ID NO:7 is the nucleotide sequence of the rat glycoprotein CD44 cDNA(GenBank Acc. No. M61875).

SEQ ID NO:8 is the predicted amino acid sequence of the translationproduct of the rat glycoprotein CD44 cDNA (SEQ ID NO:7).

SEQ ID NO:9 is the nucleotide sequence of the rat Lot-1 cDNA (GenBankAcc. No. U72620).

SEQ ID NO:10 is the predicted amino acid sequence of the translationproduct of the rat Lot-1 cDNA (SEQ ID NO:9).

SEQ ID NO:11 is the nucleotide sequence of the rat AA892598 (EST196401)cDNA. SEQ ID NO:12 is the nucleotide sequence of the rat Mrg-1 cDNA(GenBank Acc. No. AA900476).

SEQ ID NO:13 is the nucleotide sequence of the mouse ST2 cDNA (GenBankAcc. No. Y07519).

SEQ ID NO:14 is the nucleotide sequence of the mouse ST2L cDNA (GenBankAcc. No. D13695).

SEQ ID NO:15 is the nucleotide sequence of the bovine vacuolar H+-ATPasecDNA (GenBank Acc. No. M88690).

SEQ ID NO:16 is the nucleotide sequence of the human vacuolar H+-ATPasecDNA (GenBank Acc. No. NM_(—)001693).

SEQ ID NO:17 is the nucleotide sequence of the mouse vacuolar H+-ATPasecDNA (GenBank Acc. No. NM_(—)007509).

SEQ ID NO:18 is the nucleotide sequence of the human vacuolar H+-ATPasecDNA (56,000 subunit —HO57) (GenBank Acc. No. L35249).

SEQ ID NO:19 is the nucleotide sequence of the human vacuolar H+-ATPasecDNA (B subunit) (GenBank Acc. No. M60346).

SEQ ID NO:20 is the nucleotide sequence of the bovine vacuolar H+-ATPasecDNA (B subunit) (GenBank Acc. No. M83131).

SEQ ID NO:21 is the nucleotide sequence of the gallus vacuolar H+-ATPasecDNA (GenBank Acc. No. U61724).

SEQ ID NO:22 is the nucleotide sequence of the human CD44R cDNA (GenBankAcc. No. X56794).

SEQ ID NO:23 is the nucleotide sequence of the human CD44 cDNA (GenBankAcc. No. U40373).

SEQ ID NO:24 is the nucleotide sequence of the mouse CD44 cDNA (GenBankAcc. No. M27129).

SEQ ID NO:25 is the nucleotide sequence of the hamster CD44 cDNA(GenBank Acc. No. M33827).

SEQ ID NO:26 is the nucleotide sequence of the human LOT1 cDNA (GenBankAcc. No. U72621).

SEQ ID NO:27 is the nucleotide sequence of the human ZAC zinc fingerprotein cDNA (GenBank Acc. No. AJ006354).

SEQ ID NO:28 is the nucleotide sequence of the mouse ZAC1 zinc fingerprotein cDNA (GenBank Acc. No. AF147785).

SEQ ID NO:29 is the nucleotide sequence having GenBank Acc. No.AF191918.1.

SEQ ID NO:30 is the nucleotide sequence of the human putative nucleotidebinding protein, estradiol-induced (E21G3) cDNA (GenBank Acc. No.NM_(—)014366).

SEQ ID NO:31 is the nucleotide sequence of the mouse mrg-1 cDNA (GenBankAcc. No. Y15163).

SEQ ID NO:32 is the nucleotide sequence of the human p35srj cDNA(GenBank Acc. No. AF129290).

SEQ ID NO:33 is the nucleotide sequence of the human p35srj (mrg-1) cDNA(GenBank™ Acc. No. AF109161).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts by a Northern Blot the effects of 8% cyclic mechanicalstrain on the expression of Fit-1 in cultured cardiac myocytes over thecourse of time.

FIG. 2 depicts by a Northern Blot the effects of 8% cyclic mechanicalstrain, angiotensin receptor blockade, angiotensin II, IL-1b, andphorbal ester, on the expression of Fit-1 in cultured cardiac myocytesover the course of time.

FIG. 3 depicts by a Northern Blot the effects of 8% cyclic mechanicalstrain, hydrogen peroxide, and TIRON, on the expression of Fit-1 incultured cardiac myocytes over the course of time.

FIG. 4 depicts by a Northern Blot the effects of actinomycin D andcyclohexamide on the induction of Fit-1 expression during an 8% cyclicmechanical strain on cardiac myocytes over the course of time.

FIG. 5 depicts by a Northern Blot the effects of 8% cyclic mechanicalstrain alone and in combination with IL-1b, and phorbal ester in theabsence of strain, on the expression of Fit-1 in cultured cardiacmyocytes over the course of time.

FIG. 6 depicts by a Northern Blot the effects of an 8% cyclic mechanicalstrain on the expression of vacuolar ATPase in cultured cardiac myocytesover the course of time.

FIG. 7 depicts a kit embodying features of the present invention.

FIG. 8 depicts early (left) and late (right) time course of the mRNAinduction of T2/ST2 by mechanical strain in cardiac myocytes. Maximalinduction occurs at 3 hours, is sustained for 9 hours and declines by 15hours. Top panels, T1/ST2 RNA; bottom panels, ethidium bromide. No str,no strain.

FIG. 9 depicts mRNA induction of T1/ST2 by mechanical strain (8%),interleukin-1 (10 ng/ml) and phorbol ester (PMA, 200 nM) at 1 and 3hours. PMA>strain>IL-1. Top panel, T1/ST2 mRNA, bottom panel, ethidiumbromide.

FIG. 10 depicts T1/ST2 may be a gene induced by NF-κB activation duringIL-1/IL-receptor signaling in cardiac myocytes. IL-1 and strain inducedT1/ST2 mRNA in the presence of infection with control adenovirus (left).With infection of IκB adenovirus (right), which decreases NF-κB DNAbinding activity, the IL-1 induction of T1/ST2 was blocked. The straininduction of T1/ST2 was partially blocked by IκB infection suggestinganother pathway for induction of T1/ST2 by strain. Top panel, T1/ST2mRNA; bottom panel, ethidium bromide.

FIG. 11 shows expression of T1/st2 protein following myocardialinfarction in mice by immunohistochemistry at 1 day but not 3 days afterinfarction. 40× magnification.

DETAILED DESCRIPTION OF THE INVENTION

The invention involves the discovery of a number of genes that areupregulated in cardiac cells when the cells are subjected to amechanically-induced strain deformation. In view of this discovery, itis believed that the molecules of the present invention can be used isto treat cardiovascular conditions including cardiac hypertrophy,myocardial infarction, stroke, arteriosclerosis, and/or heart failure.

Additionally, methods for using these molecules in the diagnosis of anyof the foregoing cardiovascular conditions, are also provided.

Furthermore, compositions useful in the preparation of therapeuticpreparations for the treatment of the foregoing conditions, are alsoprovided.

“Upregulated,” as used herein, refers to increased expression of a geneand/or its encoded polypeptide. “Increased expression” refers toincreasing (i.e., to a detectable extent) replication, transcription,and/or translation of any of the nucleic acids of the invention (Fit-1,vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1), since upregulationof any of these processes results in concentration/amount increase ofthe polypeptide encoded by the gene (nucleic acid). Conversely,“downregulation,” or “decreased expression” as used herein, refers todecreased expression of a gene and/or its encoded polypeptide. Theupregulation or downregulation of gene expression can be directlydetermined by detecting an increase or decrease, respectively, in thelevel of mRNA for the gene, or the level of protein expression of thegene-encoded polypeptide, using any suitable means known to the art,such as nucleic acid hybridization or antibody detection methods,respectively, and in comparison to controls.

A “cardiac cell”, as used herein, refers to a cardiomyocyte.

A “molecule,” as used herein, embraces both “nucleic acids” and“polypeptides.”

“Expression,” as used herein, refers to nucleic acid and/or polypeptideexpression.

As used herein, a “subject” is a mammal or a non-human mammal In allembodiments human nucleic acids, polypeptides, and human subjects arepreferred. Although only rat sequences are exemplified in the SequenceListing and the Examples section, it is believed that the resultsobtained using such compositions are predictive of the results that maybe obtained using homologous human sequences.

In general human homologs and alleles typically will share at least 80%nucleotide identity and/or at least 85% amino acid identity to thecharacterized rat sequences of the invention. In further instances,human homologs and alleles typically will share at least 90%, 95%, oreven 99% nucleotide identity and/or at least 95%, 98%, or even 99% aminoacid identity to the characterized rat sequences, respectively. Thehomology can be calculated using various, publicly available softwaretools developed by NCBI (Bethesda, Md.). Exemplary tools include theheuristic algorithm of Altschul S F, et al., (J Mol Biol, 1990,215:403-410), also known as BLAST. Pairwise and ClustalW alignments(BLOSUM30 matrix setting) as well as Kyte-Doolittle hydropathic analysiscan be obtained using public (EMBL, Heidelberg, Germany) and commercial(e.g., the MacVector sequence analysis software from Oxford MolecularGroup/Genetics Computer Group, Madison, Wis., Accelrys, Inc., San Diego,Calif.). Watson-Crick complements of the foregoing nucleic acids alsoare embraced by the invention.

In screening for human related genes, such as homologs and alleles ofthe rat sequences described elsewhere herein, a Southern blot may beperformed using stringent conditions, together with a probe. The term“stringent conditions” as used herein refers to parameters with whichthe art is familiar. Nucleic acid hybridization parameters may be foundin references which compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. For example, stringent conditions mayrefer to hybridization at 65° C. in 6×SSC. Alternatively, stringentconditions, as used herein, may refer, for example, to hybridization at65° C. in hybridization buffer (3.5×SSC, 0.02% Ficoll, 0.02% polyvinylpyrolidone, 0.02% Bovine Serum Albumin, 2.5 mM NaH₂PO₄(pH7), 0.5% SDS, 2mM EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS issodium dodecyl sulphate; and EDTA is ethylenediaminetetra acetic acid.After hybridization, the membrane upon which the DNA is transferred iswashed at 2×SSC at room temperature and then at 0.1×SSC/0.1×SDS attemperatures up to 68° C. In a further example, an alternative to theuse of an aqueous hybridization solution is the use of a formamidehybridization solution. Stringent hybridization conditions can thus beachieved using, for example, a 50% formamide solution and 42° C.

There are other conditions, reagents, and so forth which can be used,and would result in a similar degree of stringency. The skilled artisanwill be familiar with such conditions, and thus they are not given here.It will be understood, however, that the skilled artisan will be able tomanipulate the conditions in a manner to permit the clear identificationof human homologs and alleles of the rat nucleic acids of the invention.The skilled artisan also is familiar with the methodology for screeningcells and libraries for expression of such molecules which then areroutinely isolated, followed by isolation of the pertinent nucleic acidmolecule and sequencing.

Given the teachings herein of full-length rat cDNA clones, othermammalian sequences such as the human (mouse, bovine, etc.) cDNAscorresponding to the related rat nucleic acids can be isolated from cDNAlibraries using standard colony hybridization techniques, or can beidentified using a homology search, for example, in GenBank using any ofthe algorithms described elsewhere herein. For example, sequences withGenBank Accession numbers Y07519.1 (SEQ ID NO:13) and D13695.1 (SEQ IDNO:14) for Fit-1 homologs), M88690.1 (SEQ ID NO:15), NM_(—)001693.1 (SEQID NO:16), NM_(—)007509.1 (SEQ ID NO:17), L35249.1 (SEQ ID NO:18),M60346.1 (SEQ ID NO:19), M83131.1 (SEQ ID NO:20 and U61724.1 (SEQ IDNO:21) for vacuolar ATPase homologs), X56794.1 (SEQ ID NO:22), U40373.1(SEQ ID NO:23), M27129.1 (SEQ ID NO:24), and M33827.1 (SEQ ID NO:25) forCD44 homologs), U72621.3 (SEQ ID NO:26), AJ006354.1 (SEQ ID NO:27), andAF147785.1 (SEQ ID NO:28) for Lot-1 homologs), AF191918.1 (SEQ ID NO:29)and NM_(—)014366.1 (SEQ ID NO:30) for AA892598 homologs), and Y15163.1(SEQ ID NO:31), AF129290.1 (SEQ ID NO:32), and AF109161.1 (SEQ ID NO:33)for Mrg-1 homologs), can be used interchangeably with the homologous ratsequences of the invention, in all aspects of the invention withoutdeparting from the essence of the invention.

As used herein with respect to nucleic acids, the term “isolated” means:(i) amplified in vitro by, for example, polymerase chain reaction (PCR);(ii) recombinantly produced by cloning; (iii) purified, as by cleavageand gel separation; or (iv) synthesized by, for example, chemicalsynthesis. An isolated nucleic acid is one which is readily manipulatedby recombinant DNA techniques well known in the art. Thus, a nucleotidesequence contained in a vector in which 5′ and 3′ restriction sites areknown or for which polymerase chain reaction (PCR) primer sequences havebeen disclosed is considered isolated, but a nucleic acid sequenceexisting in its native state in its natural host is not. An isolatednucleic acid may be substantially purified, but need not be. Forexample, a nucleic acid that is isolated within a cloning or expressionvector is not pure in that it may comprise only a tiny percentage of thematerial in the cell in which it resides. Such a nucleic acid isisolated, however, as the term is used herein because it is readilymanipulated by standard techniques known to those of ordinary skill inthe art.

According to the invention, expression of any of the foregoing nucleicacids (i.e., Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1),including unique fragments of the foregoing, can be determined usingdifferent methodologies. A “unique fragment,” as used herein, withrespect to a nucleic acid is one that is a “signature” for the largernucleic acid. For example, the unique fragment is long enough to assurethat its precise sequence is not found in molecules within the humangenome outside of the sequence for each nucleic acid defined above(Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1, includingtheir human alleles). Those of ordinary skill in the art may apply nomore than routine procedures to determine if a fragment is unique withinthe human genome. Unique fragments, however, exclude fragmentscompletely composed of nucleotide sequences previously published as ofthe filing date of this application.

Unique fragments can be used as probes in Southern and Northern blotassays to identify such nucleic acids, or can be used in amplificationassays such as those employing PCR. As known to those skilled in theart, large probes such as 200, 250, 300 or more nucleotides arepreferred for certain uses such as Southern and Northern blots, whilesmaller fragments will be preferred for other uses such as PCR. Uniquefragments also can be used to produce fusion proteins for generatingantibodies, or determining binding of the polypeptide fragments, or forgenerating immunoassay components. Likewise, unique fragments can beemployed to produce nonfused fragments of, for example, the Fit-1,vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1 polypeptides, useful,for example, in the preparation of antibodies, immunoassays ortherapeutic applications. Unique fragments further can be used asantisense molecules to inhibit the expression of the foregoing nucleicacids and polypeptides respectively.

As will be recognized by those skilled in the art, the size of theunique fragment will depend upon its conservancy in the genetic code.Thus, some regions of SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 12, andcomplements will require longer segments to be unique while others willrequire only short segments, typically between 12 and 32 nucleotideslong (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31 and 32 bases) or more, up to the entire length ofeach of the disclosed sequences. As mentioned above, this disclosureintends to embrace each and every fragment of each sequence, beginningat the first nucleotide, the second nucleotide and so on, up to 8nucleotides short of the end, and ending anywhere from nucleotide number8, 9, 10 and so on for each sequence, up to the very last nucleotide,(provided the sequence is unique as described above). For example,virtually any segment of the region of SEQ ID NO:1 beginning atnucleotide 1 and ending at nucleotide 2586, or SEQ ID NO:3 beginning atnucleotide 1 and ending at nucleotide 2065, or complements thereof, thatis 20 or more nucleotides in length will be unique. Those skilled in theart are well versed in methods for selecting such sequences, typicallyon the basis of the ability of the unique fragment to selectivelydistinguish the sequence of interest from other sequences in the humangenome of the fragment to those on known databases typically is all thatis necessary, although in vitro confirmatory hybridization andsequencing analysis may be performed.

As used herein with respect to polypeptides, the term “isolated” meansseparated from its native environment in sufficiently pure form so thatit can be manipulated or used for any one of the purposes of theinvention. Thus, isolated means sufficiently pure to be used (i) toraise and/or isolate antibodies, (ii) as a reagent in an assay, (iii)for sequencing, (iv) as a therapeutic, etc.

In certain aspects, the invention embraces antisense oligonucleotidesthat selectively bind to a nucleic acid molecule encoding a polypeptide,to decrease the polypeptide's activity.

As used herein, the terms “antisense molecules,” “antisenseoligonucleotide,” and “antisense” describe an oligonucleotide that is anoligoribonucleotide, oligodeoxyribonucleotide, modifiedoligoribonucleotide, or modified oligodeoxyribonucleotide whichhybridizes under physiological conditions to DNA comprising a particulargene or to an mRNA transcript of that gene and, thereby, inhibits thetranscription of that gene and/or the translation of that mRNA. Theantisense molecules are designed so as to interfere with transcriptionor translation of a target gene upon hybridization with the target geneor transcript. Those skilled in the art will recognize that the exactlength of an antisense oligonucleotide and its degree of complementaritywith its target will depend upon the specific target selected, includingthe sequence of the target and the particular bases which comprise thatsequence. It is preferred that an antisense oligonucleotide beconstructed and arranged so as to bind selectively with a target underphysiological conditions, i.e., to hybridize substantially more to thetarget sequence than to any other sequence in the target cell underphysiological conditions. Based upon SEQ ID NOs: 1, 3, 5, 7, 9, 11 and12, or upon allelic or homologous genomic and/or cDNA sequences, one ofskill in the art can easily choose and synthesize any of a number ofappropriate antisense molecules for use in accordance with the presentinvention. In order to be sufficiently selective and potent forinhibition, such antisense oligonucleotides should comprise at least 10and, more preferably, at least 15 consecutive bases which arecomplementary to the target, although in certain cases modifiedoligonucleotides as short as 7 bases in length have been usedsuccessfully as antisense oligonucleotides (Wagner et al., Nat. Med,1995, 1(11):1116-1118; Nat. Biotech., 1996, 14:840-844). Mostpreferably, the antisense oligonucleotides comprise a complementarysequence of 20-30 bases. Although oligonucleotides may be chosen whichare antisense to any region of the gene or mRNA transcripts, inpreferred embodiments the antisense oligonucleotides correspond toN-terminal or 5′ upstream sites such as translation initiation,transcription initiation or promoter sites. In addition, 3′-untranslatedregions may be targeted by antisense oligonucleotides. Targeting to mRNAsplicing sites has also been used in the art but may be less preferredif alternative mRNA splicing occurs. In addition, the antisense istargeted, preferably, to sites in which mRNA secondary structure is notexpected (see, e.g., Sainio et al., Cell Mol. Neurobiol. 14(5):439-457,1994) and at which proteins are not expected to bind. Finally, although,SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 12 disclose cDNA sequences, one ofordinary skill in the art may easily derive the genomic DNAcorresponding to the foregoing sequences. Thus, the present inventionalso provides for antisense oligonucleotides which are complementary tothe genomic DNA corresponding to SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 12.Similarly, antisense to allelic or homologous human cDNAs and genomicDNAs are enabled without undue experimentation.

In one set of embodiments, the antisense oligonucleotides of theinvention may be composed of “natural” deoxyribonucleotides,ribonucleotides, or any combination thereof. That is, the 5′ end of onenative nucleotide and the 3′ end of another native nucleotide may becovalently linked, as in natural systems, via a phosphodiesterinternucleoside linkage. These oligonucleotides may be prepared by artrecognized methods which may be carried out manually or by an automatedsynthesizer. They also may be produced recombinantly by vectors.

In preferred embodiments, however, the antisense oligonucleotides of theinvention also may include “modified” oligonucleotides. That is, theoligonucleotides may be modified in a number of ways which do notprevent them from hybridizing to their target but which enhance theirstability or targeting or which otherwise enhance their therapeuticeffectiveness.

The term “modified oligonucleotide” as used herein describes anoligonucleotide in which (1) at least two of its nucleotides arecovalently linked via a synthetic internucleoside linkage (i.e., alinkage other than a phosphodiester linkage between the 5′ end of onenucleotide and the 3′ end of another nucleotide) and/or (2) a chemicalgroup not normally associated with nucleic acids has been covalentlyattached to the oligonucleotide. Preferred synthetic internucleosidelinkages are phosphorothioates, alkylphosphonates, phosphorodithioates,phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates,carbonates, phosphate triesters, acetamidates, carboxymethyl esters andpeptides.

The term “modified oligonucleotide” also encompasses oligonucleotideswith a covalently modified base and/or sugar. For example, modifiedoligonucleotides include oligonucleotides having backbone sugars whichare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Thus modified oligonucleotides may include a2′-β-alkylated ribose group. In addition, modified oligonucleotides mayinclude sugars such as arabinose in place of ribose. The presentinvention, thus, contemplates pharmaceutical preparations containingmodified antisense molecules that are complementary to and hybridizablewith, under physiological conditions, nucleic acids encoding thepolypeptides with SEQ ID NOs: 2, 4, 6, 8, and/or 10, together withpharmaceutically acceptable carriers.

Antisense oligonucleotides may be administered as part of apharmaceutical composition. Such a pharmaceutical composition mayinclude the antisense oligonucleotides in combination with any standardphysiologically and/or pharmaceutically acceptable carriers which areknown in the art. The compositions should be sterile and contain atherapeutically effective amount of the antisense oligonucleotides in aunit of weight or volume suitable for administration to a patient. Theterm “pharmaceutically acceptable” means a non-toxic material that doesnot interfere with the effectiveness of the biological activity of theactive ingredients. The term “physiologically acceptable” refers to anon-toxic material that is compatible with a biological system such as acell, cell culture, tissue, or organism. The characteristics of thecarrier will depend on the route of administration. Physiologically andpharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art.

The invention also involves expression vectors coding for proteinsencoded by the nucleic acids corresponding to SEQ ID NOs: 1, 3, 5, 7, 9,11 and/or 12, fragments and variants thereof, and host cells containingthose expression vectors. Virtually any cells, prokaryotic oreukaryotic, which can be transformed with heterologous DNA or RNA andwhich can be grown or maintained in culture, may be used in the practiceof the invention. Examples include bacterial cells such as Escherichiacoli and mammalian cells such as mouse, hamster, pig, goat, primate,etc. They may be of a wide variety of tissue types, including mastcells, fibroblasts, oocytes and lymphocytes, and they may be primarycells or cell lines. Specific examples include CHO cells and COS cells.Cell-free transcription systems also may be used in lieu of cells.

As used herein, a “vector” may be any of a number of nucleic acids intowhich a desired sequence may be inserted by restriction and ligation fortransport between different genetic environments or for expression in ahost cell. Vectors are typically composed of DNA although RNA vectorsare also available. Vectors include, but are not limited to, plasmids,phagemids and virus genomes. A cloning vector is one which is able toreplicate in a host cell, and which is further characterized by one ormore endonuclease restriction sites at which the vector may be cut in adeterminable fashion and into which a desired DNA sequence may beligated such that the new recombinant vector retains its ability toreplicate in the host cell. In the case of plasmids, replication of thedesired sequence may occur many times as the plasmid increases in copynumber within the host bacterium or just a single time per host beforethe host reproduces by mitosis. In the case of phage, replication mayoccur actively during a lytic phase or passively during a lysogenicphase. An expression vector is one into which a desired DNA sequence maybe inserted by restriction and ligation such that it is operably joinedto regulatory sequences and may be expressed as an RNA transcript.Vectors may further contain one or more marker sequences suitable foruse in the identification of cells which have or have not beentransformed or transfected with the vector. Markers include, forexample, genes encoding proteins which increase or decrease eitherresistance or sensitivity to antibiotics or other compounds, genes whichencode enzymes whose activities are detectable by standard assays knownin the art (e.g., β-galactosidase or alkaline phosphatase), and geneswhich visibly affect the phenotype of transformed or transfected cells,hosts, colonies or plaques (e.g., green fluorescent protein). Preferredvectors are those capable of autonomous replication and expression ofthe structural gene products present in the DNA segments to which theyare operably joined.

As used herein, a coding sequence and regulatory sequences are said tobe “operably joined” when they are covalently linked in such a way as toplace the expression or transcription of the coding sequence under theinfluence or control of the regulatory sequences. If it is desired thatthe coding sequences be translated into a functional protein, two DNAsequences are said to be operably joined if induction of a promoter inthe 5′ regulatory sequences results in the transcription of the codingsequence and if the nature of the linkage between the two DNA sequencesdoes not (1) result in the introduction of a frame-shift mutation, (2)interfere with the ability of the promoter region to direct thetranscription of the coding sequences, or (3) interfere with the abilityof the corresponding RNA transcript to be translated into a protein.Thus, a promoter region would be operably joined to a coding sequence ifthe promoter region were capable of effecting transcription of that DNAsequence such that the resulting transcript might be translated into thedesired protein or polypeptide.

The precise nature of the regulatory sequences needed for geneexpression may vary between species or cell types, but shall in generalinclude, as necessary, 5′ non-transcribed and 5′ non-translatedsequences involved with the initiation of transcription and translationrespectively, such as a TATA box, capping sequence, CAAT sequence, andthe like. Such 5′ non-transcribed regulatory sequences will ofteninclude a promoter region which includes a promoter sequence fortranscriptional control of the operably joined gene. Regulatorysequences may also include enhancer sequences or upstream activatorsequences as desired. The vectors of the invention may optionallyinclude 5′ leader or signal sequences. The choice and design of anappropriate vector is within the ability and discretion of one ofordinary skill in the art.

Expression vectors containing all the necessary elements for expressionare commercially available and known to those skilled in the art. See,e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, SecondEdition, Cold Spring Harbor Laboratory Press, 1989. Cells aregenetically engineered by the introduction into the cells ofheterologous DNA (RNA) encoding a polypeptide or fragment or variantthereof. That heterologous DNA (RNA) is placed under operable control oftranscriptional elements to permit the expression of the heterologousDNA in the host cell.

Preferred systems for mRNA expression in mammalian cells are those suchas pRc/CMV (available from Invitrogen, Carlsbad, Calif.) that contain aselectable marker such as a gene that confers G418 resistance (whichfacilitates the selection of stably transfected cell lines) and thehuman cytomegalovirus (CMV) enhancer-promoter sequences. Additionally,suitable for expression in primate or canine cell lines is the pCEP4vector (Invitrogen, Carlsbad, Calif.), which contains an Epstein Barrvirus (EBV) origin of replication, facilitating the maintenance ofplasmid as a multicopy extrachromosomal element. Another expressionvector is the pEF-BOS plasmid containing the promoter of polypeptideElongation Factor 1α, which stimulates efficiently transcription invitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res.18:5322, 1990), and its use in transfection experiments is disclosed by,for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Stillanother preferred expression vector is an adenovirus, described byStratford-Perricaudet, which is defective for E1 and E3 proteins (J.Clin. Invest. 90:626-630, 1992). The use of the adenovirus as anAdeno.P1A recombinant is disclosed by Warnier et al., in intradermalinjection in mice for immunization against P1A (Int. J. Cancer,67:303-310, 1996).

The invention also embraces so-called expression kits, which allow theartisan to prepare a desired expression vector or vectors. Suchexpression kits include at least separate portions of each of thepreviously discussed coding sequences. Other components may be added, asdesired, as long as the previously mentioned sequences, which arerequired, are included.

It will also be recognized that the invention embraces the use of theabove described SEQ ID NOs: 1, 3, 5, 7, 9, 11 and/or 12 cDNAsequence-containing expression vectors, to transfect host cells and celllines, be these prokaryotic (e.g., Escherichia coli), or eukaryotic(e.g., CHO cells, COS cells, yeast expression systems and recombinantbaculovirus expression in insect cells). Especially useful are mammaliancells such as mouse, hamster, pig, goat, primate, etc. They may be of awide variety of tissue types, and include primary cells and cell lines.Specific examples include dendritic cells, U293 cells, peripheral bloodleukocytes, bone marrow stem cells and embryonic stem cells.

The invention also provides isolated polypeptides (including wholeproteins and partial proteins), encoded by the foregoing nucleic acids(SEQ ID NOs: 1, 3, 5, 7, 9, 11 and 12), and include the polypeptides ofSEQ ID NOs: 2, 4, 6, 8, and/or 10, and unique fragments thereof. Suchpolypeptides are useful, for example, alone or as part of fusionproteins to generate antibodies, as components of an immunoassay, etc.Polypeptides can be isolated from biological samples including tissue orcell homogenates, and can also be expressed recombinantly in a varietyof prokaryotic and eukaryotic expression systems by constructing anexpression vector appropriate to the expression system, introducing theexpression vector into the expression system, and isolating therecombinantly expressed protein. Short polypeptides, including antigenicpeptides (such as are presented by MHC molecules on the surface of acell for immune recognition) also can be synthesized chemically usingwell-established methods of peptide synthesis.

A unique fragment for each of the foregoing polypeptide, in general, hasthe features and characteristics of unique fragments as discussed abovein connection with nucleic acids. As will be recognized by those skilledin the art, the size of the unique fragment will depend upon factorssuch as whether the fragment constitutes a portion of a conservedprotein domain. Thus, some regions of a polypeptide will require longersegments to be unique while others will require only short segments,typically between 5 and 12 amino acids (e.g. 5, 6, 7, 8, 9, 10, 11 and12 amino acids long or more, including each integer up to the fulllength of each polypeptide).

Unique fragments of a polypeptide preferably are those fragments whichretain a distinct functional capability of the polypeptide. Functionalcapabilities which can be retained in a unique fragment of a polypeptideinclude interaction with antibodies, interaction with other polypeptidesor fragments thereof, interaction with other molecules, etc. Oneimportant activity is the ability to act as a signature for identifyingthe polypeptide. Those skilled in the art are well versed in methods forselecting unique amino acid sequences, typically on the basis of theability of the unique fragment to selectively distinguish the sequenceof interest from non-family members. A comparison of the sequence of thefragment to those on known databases typically is all that is necessary.

The invention embraces variants of the polypeptides described above. Asused herein, a “variant” of a polypeptide is a polypeptide whichcontains one or more modifications to the primary amino acid sequence ofa natural (e.g., “wild-type”: a polypeptide with an amino acid sequenceselected from the group consisting of SEQ ID NO: 2, 4, 6, 8, and 10)polypeptide. Modifications which create a polypeptide variant aretypically made to the nucleic acid which encodes the polypeptide, andcan include deletions, point mutations, truncations, amino acidsubstitutions and addition of amino acids or non-amino acid moieties to:(1) reduce or eliminate an activity of a polypeptide; (2) enhance aproperty of a polypeptide, such as protein stability in an expressionsystem or the stability of protein-ligand binding; (3) provide a novelactivity or property to a polypeptide, such as addition of an antigenicepitope or addition of a detectable moiety; or (4) to provide equivalentor better binding to a polypeptide receptor or other molecule.Alternatively, modifications can be made directly to the polypeptide,such as by cleavage, addition of a linker molecule, addition of adetectable moiety, such as biotin, addition of a fatty acid, and thelike. Modifications also embrace fusion proteins comprising all or partof the polypeptide's amino acid sequence. One of skill in the art willbe familiar with methods for predicting the effect on proteinconformation of a change in protein sequence, and can thus “design” avariant polypeptide according to known methods. One example of such amethod is described by Dahiyat and Mayo in Science 278:82-87, 1997,whereby proteins can be designed de novo. The method can be applied to aknown protein to vary only a portion of the polypeptide sequence. Byapplying the computational methods of Dahiyat and Mayo, specificvariants of any of the foregoing polypeptides can be proposed and testedto determine whether the variant retains a desired conformation.

Variants can include polypeptides which are modified specifically toalter a feature of the polypeptide unrelated to its physiologicalactivity. For example, cysteine residues can be substituted or deletedto prevent unwanted disulfide linkages. Similarly, certain amino acidscan be changed to enhance expression of a polypeptide by eliminatingproteolysis by proteases in an expression system (e.g., dibasic aminoacid residues in yeast expression systems in which KEX2 proteaseactivity is present).

Mutations of a nucleic acid which encodes a polypeptide preferablypreserve the amino acid reading frame of the coding sequence, andpreferably do not create regions in the nucleic acid which are likely tohybridize to form secondary structures, such a hairpins or loops, whichcan be deleterious to expression of the variant polypeptide.

Mutations can be made by selecting an amino acid substitution, or byrandom mutagenesis of a selected site in a nucleic acid which encodesthe polypeptide. Variant polypeptides are then expressed and tested forone or more activities to determine which mutation provides a variantpolypeptide with the desired properties. Further mutations can be madeto variants (or to non-variant polypeptides) which are silent as to theamino acid sequence of the polypeptide, but which provide preferredcodons for translation in a particular host. The preferred codons fortranslation of a nucleic acid in, e.g., Escherichia coli, are well knownto those of ordinary skill in the art. Still other mutations can be madeto the noncoding sequences of a gene or cDNA clone to enhance expressionof the polypeptide.

The skilled artisan will realize that conservative amino acidsubstitutions may be made in any of the foregoing polypeptides toprovide functionally equivalent variants of the foregoing polypeptides,i.e., the variants retain the functional capabilities of eachpolypeptide. As used herein, a “conservative amino acid substitution”refers to an amino acid substitution which does not significantly alterthe tertiary structure and/or activity of the polypeptide. Variants canbe prepared according to methods for altering polypeptide sequence knownto one of ordinary skill in the art, and include those that are found inreferences which compile such methods, e.g. Molecular Cloning: ALaboratory Manual, J. Sambrook, et al., eds., Second Edition, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, orCurrent Protocols in Molecular Biology, F. M. Ausubel, et al., eds.,John Wiley & Sons, Inc., New York. Conservative substitutions of aminoacids include substitutions made amongst amino acids within thefollowing groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G;(e) S, T; (f) Q, N; and (g) E, D.

Thus functionally equivalent variants of polypeptides, i.e., variants ofpolypeptides which retain the function of the natural (“wild-type”)polypeptides, are contemplated by the invention. Conservative amino acidsubstitutions in the amino acid sequence of polypeptides to producefunctionally equivalent variants of each polypeptide typically are madeby alteration of a nucleic acid encoding the polypeptide. Suchsubstitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis according tothe method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488-492,1985), or by chemical synthesis of a gene encoding a polypeptide. Theactivity of functionally equivalent fragments of polypeptides can betested by cloning the gene encoding the altered polypeptide into abacterial or mammalian expression vector, introducing the vector into anappropriate host cell, expressing the altered polypeptide, and testingfor a functional capability of the polypeptides as disclosed herein

The invention as described herein has a number of uses, some of whichare described elsewhere herein. First, the invention permits isolationof polypeptides. A variety of methodologies well-known to the skilledartisan can be utilized to obtain isolated molecules. The polypeptidemay be purified from cells which naturally produce the polypeptide bychromatographic means or immunological recognition. Alternatively, anexpression vector may be introduced into cells to cause production ofthe polypeptide. In another method, mRNA transcripts may bemicroinjected or otherwise introduced into cells to cause production ofthe encoded polypeptide. Translation of mRNA in cell-free extracts suchas the reticulocyte lysate system also may be used to producepolypeptides. Those skilled in the art also can readily follow knownmethods for isolating polypeptides. These include, but are not limitedto, immunochromatography, HPLC, size-exclusion chromatography,ion-exchange chromatography and immune-affinity chromatography.

The invention also provides, in certain embodiments, “dominant negative”polypeptides derived from polypeptides. A dominant negative polypeptideis an inactive variant of a protein, which, by interacting with thecellular machinery, displaces an active protein from its interactionwith the cellular machinery or competes with the active protein, therebyreducing the effect of the active protein. For example, a dominantnegative receptor which binds a ligand but does not transmit a signal inresponse to binding of the ligand can reduce the biological effect ofexpression of the ligand. Likewise, a dominant negativecatalytically-inactive kinase which interacts normally with targetproteins but does not phosphorylate the target proteins can reducephosphorylation of the target proteins in response to a cellular signal.Similarly, a dominant negative transcription factor which binds to apromoter site in the control region of a gene but does not increase genetranscription can reduce the effect of a normal transcription factor byoccupying promoter binding sites without increasing transcription.

The end result of the expression of a dominant negative polypeptide in acell is a reduction in function of active proteins. One of ordinaryskill in the art can assess the potential for a dominant negativevariant of a protein, and use standard mutagenesis techniques to createone or more dominant negative variant polypeptides. See, e.g., U.S. Pat.No. 5,580,723 and Sambrook et al., Molecular Cloning: A LaboratoryManual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Theskilled artisan then can test the population of mutagenized polypeptidesfor diminution in a selected activity and/or for retention of such anactivity. Other similar methods for creating and testing dominantnegative variants of a protein will be apparent to one of ordinary skillin the art.

The isolation of the cDNAs of the invention also makes it possible forthe artisan to diagnose a disorder characterized by an aberrantexpression of any of the foregoing cDNAs. These methods involvedetermining expression of each of the identified nucleic acids, and/orpolypeptides derived therefrom. In the former situation, suchdeterminations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction, orassaying with labeled hybridization probes as exemplified below. In thelatter situation, such determination can be carried out via any standardimmunological assay using, for example, antibodies which bind to thesecreted protein.

The invention also embraces isolated peptide binding agents which, forexample, can be antibodies or fragments of antibodies (“bindingpolypeptides”), having the ability to selectively bind to any of thepolypeptides of the invention (e.g., SEQ ID NO: 2, 4, 6, 8, or 10).Antibodies include polyclonal and monoclonal antibodies, preparedaccording to conventional methodology.

Significantly, as is well-known in the art, only a small portion of anantibody molecule, the paratope, is involved in the binding of theantibody to its epitope (see, in general, Clark, W. R. (1986) TheExperimental Foundations of Modern Immunology Wiley & Sons, Inc., NewYork; Roitt, I. (1991) Essential Immunology, 7th Ed., BlackwellScientific Publications, Oxford). The pFc′ and Fc regions, for example,are effectors of the complement cascade but are not involved in antigenbinding. An antibody from which the pFc′ region has been enzymaticallycleaved, or which has been produced without the pFc′ region, designatedan F(ab′)₂ fragment, retains both of the antigen binding sites of anintact antibody. Similarly, an antibody from which the Fc region hasbeen enzymatically cleaved, or which has been produced without the Fcregion, designated an Fab fragment, retains one of the antigen bindingsites of an intact antibody molecule. Proceeding further, Fab fragmentsconsist of a covalently bound antibody light chain and a portion of theantibody heavy chain denoted Fd. The Fd fragments are the majordeterminant of antibody specificity (a single Fd fragment may beassociated with up to ten different light chains without alteringantibody specificity) and Fd fragments retain epitope-binding ability inisolation.

Within the antigen-binding portion of an antibody, as is well-known inthe art, there are complementarity determining regions (CDRs), whichdirectly interact with the epitope of the antigen, and framework regions(FRs), which maintain the tertiary structure of the paratope (see, ingeneral, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragmentand the light chain of IgG immunoglobulins, there are four frameworkregions (FR1 through FR4) separated respectively by threecomplementarity determining regions (CDR1 through CDR3). The CDRs, andin particular the CDR3 regions, and more particularly the heavy chainCDR3, are largely responsible for antibody specificity.

It is now well-established in the art that the non-CDR regions of amammalian antibody may be replaced with similar regions of conspecificor heterospecific antibodies while retaining the epitopic specificity ofthe original antibody. This is most clearly manifested in thedevelopment and use of “humanized” antibodies in which non-human CDRsare covalently joined to human FR and/or Fc/pFc′ regions to produce afunctional antibody. See, e.g., U.S. Pat. Nos. 4,816,567; 5,225,539;5,585,089; 5,693,762 and 5,859,205. Thus, for example, PCT InternationalPublication Number WO 92/04381 teaches the production and use ofhumanized murine RSV antibodies in which at least a portion of themurine FR regions have been replaced by FR regions of human origin. Suchantibodies, including fragments of intact antibodies withantigen-binding ability, are often referred to as “chimeric” antibodies.

Thus, as will be apparent to one of ordinary skill in the art, thepresent invention also provides for F(ab′)₂, Fab, Fv and Fd fragments;chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2and/or light chain CDR3 regions have been replaced by is homologoushuman or non-human sequences; chimeric F(ab′)₂ fragment antibodies inwhich the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regionshave been replaced by homologous human or non-human sequences; chimericFab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/orlight chain CDR3 regions have been replaced by homologous human ornon-human sequences; and chimeric Fd fragment antibodies in which the FRand/or CDR1 and/or CDR2 regions have been replaced by homologous humanor non-human sequences. The present invention also includes so-calledsingle chain antibodies.

Thus, the invention involves polypeptides of numerous size and type thatbind specifically to polypeptides of the invention (e.g., SEQ ID NO: 2,4, 6, 8, or 10), and complexes of both the polypeptides and theirbinding partners. These polypeptides may be derived also from sourcesother than antibody technology. For example, such polypeptide bindingagents can be provided by degenerate peptide libraries which can bereadily prepared in solution, in immobilized form, as bacterial flagellapeptide display libraries or as phage display libraries. Combinatoriallibraries also can be synthesized of peptides containing one or moreamino acids. Libraries further can be synthesized of peptides andnon-peptide synthetic moieties.

The invention further provides efficient methods of identifying agentsor lead compounds for agents active at the level of a polypeptide orpolypeptide fragment dependent cellular function. In particular, suchfunctions include interaction with other polypeptides or fragments.Generally, the screening methods involve assaying for compounds whichinterfere with the activity of a polypeptide of the invention, althoughcompounds which enhance such activity also can be assayed using thescreening methods. Such methods are adaptable to automated, highthroughput screening of compounds. Target indications include cellularprocesses modulated by such polypeptides, for example, overexpression incells under mechanical strains.

A wide variety of assays for candidate (pharmacological) agents areprovided, including, labeled in vitro protein-ligand binding assays,electrophoretic mobility shift assays, immunoassays, cell-based assayssuch as two- or three-hybrid screens, expression assays, etc. Thetransfected nucleic acids can encode, for example, combinatorial peptidelibraries or cDNA libraries. Convenient reagents for such assays, e.g.,GAL4 fusion proteins, are known in the art. An exemplary cell-basedassay involves transfecting a cell with a nucleic acid encoding apolypeptide of the invention fused to a GAL4 DNA binding domain and anucleic acid encoding a reporter gene operably joined to a geneexpression regulatory region, such as one or more GAL4 binding sites.Activation of reporter gene transcription occurs when the reporterfusion polypeptide binds an agent such as to enable transcription of thereporter gene. Agents which modulate polypeptide mediated cell functionare then detected through a change in the expression of reporter gene.Methods for determining changes in the expression of a reporter gene areknown in the art.

Polypeptide fragments used in the methods, when not produced by atransfected nucleic acid are added to an assay mixture as an isolatedpolypeptide. Polypeptides preferably are produced recombinantly,although such polypeptides may be isolated from biological extracts.Recombinantly produced polypeptides include chimeric proteins comprisinga fusion of a protein of the invention with another polypeptide, e.g., apolypeptide capable of providing or enhancing protein-protein binding,sequence specific nucleic acid binding (such as GAL4), enhancingstability of the polypeptide of the invention under assay conditions, orproviding a detectable moiety, such as green fluorescent protein or aFlag epitope.

The assay mixture is comprised of a natural intracellular orextracellular binding target capable of interacting with a polypeptideof the invention. While natural polypeptide binding targets may be used,it is frequently preferred to use portions (e.g., peptides or nucleicacid fragments) or analogs (i.e., agents which mimic the polypeptide'sbinding properties of the natural binding target for purposes of theassay) of the polypeptide binding target so long as the portion oranalog provides binding affinity and avidity to the polypeptide fragmentmeasurable in the assay.

The assay mixture also comprises a candidate agent. Typically, aplurality of assay mixtures are run in parallel with different agentconcentrations to obtain a different response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e., at zero concentration of agent or at aconcentration of agent below the limits of assay detection. Candidateagents encompass numerous chemical classes, although typically they areorganic compounds. Preferably, the candidate agents are small organiccompounds, i.e., those having a molecular weight of more than about 50yet less than about 2500, preferably less than about 1000 and, morepreferably, less than about 500. Candidate agents comprise functionalchemical groups necessary for structural interactions with polypeptidesand/or nucleic acids, and typically include at least an amine, carbonyl,hydroxyl or carboxyl group, preferably at least two of the functionalchemical groups and more preferably at least three of the functionalchemical groups. The candidate agents can comprise cyclic carbon orheterocyclic structure and/or aromatic or polyaromatic structuressubstituted with one or more of the above-identified functional groups.Candidate agents also can be biomolecules such as peptides, saccharides,fatty acids, sterols, isoprenoids, purines, pyrimidines, derivatives orstructural analogs of the above, or combinations thereof and the like.Where the agent is a nucleic acid, the agent typically is a DNA or RNAmolecule, although modified nucleic acids as defined herein are alsocontemplated.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random peptides, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plantand animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bemodified through conventional chemical, physical, and biochemical means.Further, known (pharmacological) agents may be subjected to directed orrandom chemical modifications such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs of theagents.

A variety of other reagents also can be included in the mixture. Theseinclude reagents such as salts, buffers, neutral proteins (e.g.,albumin), detergents, etc. which may be used to facilitate optimalprotein-protein and/or protein-nucleic acid binding. Such a reagent mayalso reduce non-specific or background interactions of the reactioncomponents. Other reagents that improve the efficiency of the assay suchas protease inhibitors, nuclease inhibitors, antimicrobial agents, andthe like may also be used.

The mixture of the foregoing assay materials is incubated underconditions whereby, but for the presence of the candidate agent, thechosen polypeptide of the invention specifically binds a cellularbinding target, a portion thereof or analog thereof. The order ofaddition of components, incubation temperature, time of incubation, andother parameters of the assay may be readily determined. Suchexperimentation merely involves optimization of the assay parameters,not the fundamental composition of the assay. Incubation temperaturestypically are between 4° C. and 40° C. Incubation times preferably areminimized to facilitate rapid, high throughput screening, and typicallyare between 0.1 and 10 hours.

After incubation, the presence or absence of specific binding betweenthe polypeptide and one or more binding targets is detected by anyconvenient method available to the user. For cell free binding typeassays, a separation step is often used to separate bound from unboundcomponents. The separation step may be accomplished in a variety ofways. Conveniently, at least one of the components is immobilized on asolid substrate, from which the unbound components may be easilyseparated. The solid substrate can be made of a wide variety ofmaterials and in a wide variety of shapes, e.g., microtiter plate,microbead, dipstick, resin particle, etc. The substrate preferably ischosen to maximize signal to noise ratios, primarily to minimizebackground binding, as well as for ease of separation and cost.

Separation may be effected for example, by removing a bead or dipstickfrom a reservoir, emptying or diluting a reservoir such as a microtiterplate well, rinsing a bead, particle, chromatograpic column or filterwith a wash solution or solvent. The separation step preferably includesmultiple rinses or washes. For example, when the solid substrate is amicrotiter plate, the wells may be washed several times with a washingsolution, which typically includes those components of the incubationmixture that do not participate in specific bindings such as salts,buffer, detergent, a non-specific protein, etc. When the solid substrateis a magnetic bead(s), the bead(s) may be washed one or more times witha washing solution and isolated using a magnet.

Detection may be effected in any convenient way for cell-based assayssuch as two- or three-hybrid screens. The transcript resulting from areporter gene transcription assay of a polypeptide interacting with atarget molecule typically encodes a directly or indirectly detectableproduct, e.g., β-galactosidase activity, luciferase activity, and thelike. For cell free binding assays, one of the components usuallycomprises, or is coupled to, a detectable label. A wide variety oflabels can be used, such as those that provide direct detection (e.g.,radioactivity, luminescence, optical or electron density, etc), orindirect detection (e.g., epitope tag such as the FLAG epitope, enzymetag such as horseseradish peroxidase, etc.). The label may be bound to abinding partner of the polypeptide, or incorporated into the structureof the binding partner.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,streptavidin-biotin conjugates, etc. Methods for detecting the labelsare well known in the art.

The invention provides polypeptide-specific binding agents, methods ofidentifying and making such agents, and their use in diagnosis, therapyand pharmaceutical development. For example, polypeptide-specificpharmacological agents are useful in a variety of diagnostic andtherapeutic applications, especially where disease or disease prognosisis associated with altered polypeptide binding characteristics. Novelpolypeptide-specific binding agents include polypeptide-specificantibodies, cell surface receptors, and other natural intracellular andextracellular binding agents identified with assays such as two hybridscreens, and non-natural intracellular and extracellular binding agentsidentified in screens of chemical libraries and the like.

In general, the specificity of polypeptide binding to a specificmolecule is determined by binding equilibrium constants. Targets whichare capable of selectively binding a polypeptide preferably have bindingequilibrium constants of at least about 10⁷ M⁻¹, more preferably atleast about 10⁸ M⁻¹, and most preferably at least about 10⁹ M⁻¹. A widevariety of cell based and cell free assays may be used to demonstratepolypeptide-specific binding. Cell based assays include one, two andthree hybrid screens, assays in which polypeptide-mediated transcriptionis inhibited or increased, etc. Cell free assays include protein bindingassays, immunoassays, etc. Other assays useful for screening agentswhich bind polypeptides of the invention include fluorescence resonanceenergy transfer (FRET), and electrophoretic mobility shift analysis(EMSA).

According to still another aspect of the invention, a method ofdiagnosing a disorder characterized by aberrant expression of a nucleicacid molecule, an expression product thereof, or a fragment of anexpression product thereof, is provided. The method involves contactinga biological sample isolated from a subject with an agent thatspecifically binds to the nucleic acid molecule, an expression productthereof, or a fragment of an expression product thereof, and determiningthe interaction between the agent and the nucleic acid molecule or theexpression product as a determination of the disorder, wherein thenucleic acid molecule is selected from the group consisting of Fit-1,vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1. In some embodiments,the disorder is a cardiovascular condition selected from the groupconsisting of myocardial infarction, stroke, arteriosclerosis, and heartfailure. In one embodiment, the disorder is cardiac hypertrophy.

In the case where the molecule is a nucleic acid molecule, suchdeterminations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction, orassaying with labeled hybridization probes as exemplified herein. In thecase is where the molecule is an expression product of the nucleic acidmolecule, or a fragment of an expression product of the nucleic acidmolecule, such determination can be carried out via any standardimmunological assay using, for example, antibodies which bind to any ofthe polypeptide expression products.

“Aberrant expression” refers to decreased expression (underexpression)or increased expression (overexpression) of any of the foregoingmolecules (Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1,nucleic acids and/or polypeptides) in comparison with a control (i.e.,expression of the same molecule in a healthy or “normal” subject). A“healthy subject,” as used herein, refers to a subject who is not atrisk for developing a future cardiovascular condition (see earlierdiscussion and Harrison's Principles of Experimental Medicine, 13thEdition, McGraw-Hill, Inc., N.Y.—hereinafter “Harrison's”). Healthysubjects also do not otherwise exhibit symptoms of disease. In otherwords, such subjects, if examined by a medical professional, would becharacterized as healthy and free of symptoms of a cardiovasculardisorder or at risk of developing a cardiovascular disorder.

When the disorder is a cardiovascular condition selected from the groupconsisting of cardiac hypertrophy, myocardial infarction, stroke,arteriosclerosis, and heart failure, decreased expression of any of theforegoing molecules in comparison with a control (e.g., a healthyindividual) is indicative of the presence of the disorder, or indicativeof the risk for developing such disorder in the future.

The invention also provides novel kits which could be used to measurethe levels of the nucleic acids of the invention, or expression productsof the invention.

In one embodiment, a kit comprises a package containing an agent thatselectively binds to an isolated nucleic acid selected from the groupconsisting of Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1,or expression products thereof, and a control for comparing to ameasured value of binding of said agent any of the foregoing isolatednucleic acids or expression products thereof. Kits are generallycomprised of the following major elements: packaging, an agent of theinvention, a control agent, and instructions. Packaging may be abox-like structure for holding a vial (or number of vials) containing anagent of the invention, a vial (or number of vials) containing a controlagent, and instructions. Individuals skilled in the art can readilymodify the packaging to suit individual needs. In some embodiments, thecontrol is a predetermined value for comparing to the measured value. Incertain embodiments, the control comprises an epitope of the expressionproduct of any of the foregoing isolated nucleic acids.

In the case of nucleic acid detection, pairs of primers for amplifying anucleic acid molecule of the invention can be included. The preferredkits would include controls such as known amounts of nucleic acidprobes, epitopes (such as Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598,and Mrg-1 expression products) or anti-epitope antibodies, as well asinstructions or other printed material. In certain embodiments theprinted material can characterize risk of developing a cardiovascularcondition based upon the outcome of the assay. The reagents may bepackaged in containers and/or coated on wells in predetermined amounts,and the kits may include standard materials such as labeledimmunological reagents (such as labeled anti-IgG antibodies) and thelike. One kit is a packaged polystyrene microtiter plate coated with anyof the foregoing proteins of the invention and a container containinglabeled anti-human IgG antibodies. A well of the plate is contactedwith, for example, a biological fluid, washed and then contacted withthe anti-IgG antibody. The label is then detected. A kit embodyingfeatures of the present invention, generally designated by the numeral11, is illustrated in FIG. 7. Kit 11 is comprised of the following majorelements: packaging 15, an agent of the invention 17, a control agent19, and instructions 21. Packaging 15 is a box-like structure forholding a vial (or number of vials) containing an agent of the invention17, a vial (or number of vials) containing a control agent 19, andinstructions 21. Individuals skilled in the art can readily modifypackaging 15 to suit individual needs.

The invention also embraces methods for treating a cardiovascularcondition. In some embodiments, the method involves administering to asubject in need of such treatment a molecule selected from the groupconsisting of Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1,in an amount effective to treat the cardiovascular condition. In certainembodiments, the method involves administering to a subject in need ofsuch treatment an agent that increases expression of any of theforegoing molecules (Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, andMrg-1), in an amount effective to treat the cardiovascular condition.

“Agents that increase expression” of a nucleic acid or a polypeptide, asused herein, are known in the art, and refer to sense nucleic acids,polypeptides encoded by the nucleic acids, and other agents that enhanceexpression of such molecules (e.g., transcription factors specific forthe nucleic acids that enhance their expression). Any agents thatincrease expression of a molecule (and as described herein, increase itsactivity), are useful according to the invention.

In certain embodiments, the molecule is a nucleic acid. In someembodiments the nucleic acid is operatively coupled to a gene expressionsequence which directs the expression of the nucleic acid moleculewithin a cardiomyocyte. The “gene expression sequence” is any regulatorynucleotide sequence, such as a promoter sequence or promoter-enhancercombination, which facilitates the efficient transcription andtranslation of the nucleic acid to which it is operably joined. The geneexpression sequence may, for example, be a mammalian or viral promoter,such as a constitutive or inducible promoter. Constitutive mammalianpromoters include, but are not limited to, the promoters for thefollowing genes: hypoxanthine phosphoribosyl transferase (HPTR),adenosine deaminase, pyruvate kinase, α-actin promoter and otherconstitutive promoters. Exemplary viral promoters which functionconstitutively in eukaryotic cells include, for example, promoters fromthe simian virus, papilloma virus, adenovirus, human immunodeficiencyvirus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminalrepeats (LTR) of Moloney leukemia virus and other retroviruses, and thethymidine kinase promoter of herpes simplex virus. Other constitutivepromoters are known to those of ordinary skill in the art. The promotersuseful as gene expression sequences of the invention also includeinducible promoters. Inducible promoters are activated in the presenceof an inducing agent. For example, the metallothionein promoter isactivated to increase transcription and translation in the presence ofcertain metal ions. Other inducible promoters are known to those ofordinary skill in the art.

In general, the gene expression sequence shall include, as necessary, 5′non-transcribing and 5′ non-translating sequences involved with theinitiation of transcription and translation, respectively, such as aTATA box, capping sequence, CAAT sequence, and the like. Especially,such 5′ non-transcribing sequences will include a promoter region whichincludes a promoter sequence for transcriptional control of the operablyjoined nucleic acid. The gene expression sequences optionally includesenhancer sequences or upstream activator sequences as desired.

Preferably, any of the nucleic acid molecules of the invention (e.g.,Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1) is linked to agene expression sequence which permits expression of the nucleic acidmolecule in a cell such as a cardiomyocyte and/or a vascular endothelialcell (including a smooth muscle cell). More preferably, the geneexpression sequence permits expression of the nucleic acid molecule in acardiomyocyte, and does not permit expression of the molecule in a cellselected from the group consisting of a neuronal cell, a fibroblast, anda cell of hematopoietic origin. A sequence which permits expression ofthe nucleic acid molecule in a cardiomyocyte, is one which isselectively active in such a cell type, thereby causing expression ofthe nucleic acid molecule in the cell. The cardiac myosin heavy chaingene promoter, for example, can be used to express any of the foregoingnucleic acid molecules of the invention in a cardiomyocyte. Those ofordinary skill in the art will be able to easily identify alternativepromoters that are capable of expressing a nucleic acid molecule in acardiomyocyte.

The nucleic acid sequence and the gene expression sequence are said tobe “operably joined” when they are covalently linked in such a way as toplace the transcription and/or translation of the nucleic acid codingsequence under the influence or control of the gene expression sequence.If it is desired that the nucleic acid sequence be translated into afunctional protein, two DNA sequences are said to be operably joined ifinduction of a promoter in the 5′ gene expression sequence results inthe transcription of the nucleic acid sequence and if the nature of thelinkage between the two DNA sequences does not (1) result in theintroduction of a frame-shift mutation, (2) interfere with the abilityof the promoter region to direct the transcription of the nucleic acidsequence, and/or (3) interfere with the ability of the corresponding RNAtranscript to be translated into a protein. Thus, a gene expressionsequence would be operably linked to a nucleic acid sequence if the geneexpression sequence were capable of effecting transcription of thatnucleic acid sequence such that the resulting transcript might betranslated into the desired protein or polypeptide.

The molecules of the invention can be delivered to the preferred celltypes of the invention alone or in association with a vector. In itsbroadest sense, a “vector” is any vehicle capable of facilitating: (1)delivery of a molecule to a target cell and/or (2) uptake of themolecule by a target cell. Preferably, the vectors transport themolecule into the target cell with reduced degradation relative to theextent of degradation that would result in the absence of the vector.Optionally, a “targeting ligand” can be attached to the vector toselectively deliver the vector to a cell which expresses on its surfacethe cognate receptor for the targeting ligand. In this manner, thevector (containing a nucleic acid or a protein) can be selectivelydelivered to a cardiomyocyte cell in, e.g., the myocardium.Methodologies for targeting include conjugates, such as those describedin U.S. Pat. No. 5,391,723 to Priest. Another example of a well-knowntargeting vehicle is a liposome. Liposomes are commercially availablefrom Gibco BRL (Life Technologies Inc., Rockville, Md.). Numerousmethods are published for making targeted liposomes. Preferably, themolecules of the invention are targeted for delivery to cardiomyocytes,and/or vascular endothelial cells.

In general, the vectors useful in the invention include, but are notlimited to, plasmids, phagemids, viruses, other vehicles derived fromviral or bacterial sources that have been manipulated by the insertionor incorporation of the nucleic acid sequences of the invention, andadditional nucleic acid fragments (e.g., enhancers, promoters) which canbe attached to the nucleic acid sequences of the invention. Viralvectors are a preferred type of vector and include, but are not limitedto, nucleic acid sequences from the following viruses: adenovirus;adeno-associated virus; retrovirus, such as Moloney murine leukemiavirus; Harvey murine sarcoma virus; murine mammary tumor virus; rousesarcoma virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses;papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNAviruses such as a retrovirus. One can readily employ other vectors notnamed but known in the art.

A particularly preferred virus for certain applications is theadeno-associated virus, a double-stranded DNA virus. Theadeno-associated virus is capable of infecting a wide range of celltypes and species and can be engineered to be replication-deficienti.e., capable of directing synthesis of the desired proteins, butincapable of manufacturing an infectious particle. It further hasadvantages, such as heat and lipid solvent stability, high transductionfrequencies in cells of diverse lineages, including hematopoietic cells,and lack of superinfection inhibition thus allowing multiple series oftransductions. Reportedly, the adeno-associated virus can integrate intohuman cellular DNA in a site-specific manner, thereby minimizing thepossibility of insertional mutagenesis and variability of inserted geneexpression. In addition, wild-type adeno-associated virus infectionshave been followed in tissue culture for greater than 100 passages inthe absence of selective pressure, implying that the adeno-associatedvirus genomic integration is a relatively stable event. Theadeno-associated virus can also function in an extrachromosomal fashion.

In general, other preferred viral vectors are based on non-cytopathiceukaryotic viruses in which non-essential genes have been replaced withthe gene of interest. Non-cytopathic viruses include retroviruses, thelife cycle of which involves reverse transcription of genomic viral RNAinto DNA with subsequent proviral integration into host cellular DNA.Adenoviruses and retroviruses have been approved for human gene therapytrials. In general, the retroviruses are replication-deficient. Suchgenetically altered retroviral expression vectors have general utilityfor the high-efficiency transduction of genes in vivo. Standardprotocols for producing replication-deficient retroviruses (includingthe steps of incorporation of exogenous genetic material into a plasmid,transfection of a packaging cell line with plasmid, production ofrecombinant retroviruses by the packaging cell line, collection of viralparticles from tissue culture media, and infection of the target cellswith viral particles) are provided in Kriegler, M., “Gene Transfer andExpression, A Laboratory Manual,” W.H. Freeman C.O., New York (1990) andMurry, E. J. Ed. “Methods in Molecular Biology,” vol. 7, Humana Press,Inc., Cliffton, N.J. (1991).

Another preferred retroviral vector is the vector derived from theMoloney murine leukemia virus, as described in Nabel, E. G., et al.,Science, 1990, 249:1285-1288. These vectors reportedly were effectivefor the delivery of genes to all three layers of the arterial wall,including the media. Other preferred vectors are disclosed in Flugelman,et al., Circulation, 1992, 85:1110-1117. Additional vectors that areuseful for delivering molecules of the invention are described in U.S.Pat. No. 5,674,722 by Mulligan, et. al.

In addition to the foregoing vectors, other delivery methods may be usedto deliver a molecule of the invention to a cell such as a cardiomyocyteand/or a vascular endothelial cell, and facilitate uptake thereby.

A preferred such delivery method of the invention is a colloidaldispersion system. Colloidal dispersion systems include lipid-basedsystems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. A preferred colloidal system of the invention is a liposome.Liposomes are artificial membrane vessels which are useful as a deliveryvector in vivo or in vitro. It has been shown that large unilamellarvessels (LUV), which range in size from 0.2-4.0 μm can encapsulate largemacromolecules. RNA, DNA, and intact virions can be encapsulated withinthe aqueous interior and be delivered to cells in a biologically activeform (Fraley, et al., Trends Biochem. Sci., 1981, 6:77). In order for aliposome to be an efficient gene transfer vector, one or more of thefollowing characteristics should be present: (1) encapsulation of thegene of interest at high efficiency with retention of biologicalactivity; (2) preferential and substantial binding to a target cell incomparison to non-target cells; (3) delivery of the aqueous contents ofthe vesicle to the target cell cytoplasm at high efficiency; and (4)accurate and effective expression of genetic information.

Liposomes may be targeted to a particular tissue, such as the myocardiumor the vascular cell wall, by coupling the liposome to a specific ligandsuch as a monoclonal antibody, sugar, glycolipid, or protein. Ligandswhich may be useful for targeting a liposome to the vascular wallinclude, but are not limited to, the viral coat protein of theHemagglutinating virus of Japan. Additionally, the vector may be coupledto a nuclear targeting peptide, which will direct the nucleic acid tothe nucleus of the host cell.

Liposomes are commercially available from Gibco BRL, for example, asLIPOFECTINT™ and LIPOFECTACE™, which are formed of cationic lipids suchas N-[1-(2,3-dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride(DOTMA) and dimethyl dioctadecylammonium bromide (DDAB). Methods formaking liposomes are well known in the art and have been described inmany publications. Liposomes also have been reviewed by Gregoriadis, G.in Trends in Biotechnology, V. 3, p. 235-241 (1985). Novel liposomes forthe intracellular delivery of macromolecules, including nucleic acids,are also described in PCT International application no. PCT/US96/07572(Publication No. WO 96/40060, entitled “Intracellular Delivery ofMacromolecules”).

In one particular embodiment, the preferred vehicle is a biocompatiblemicro particle or implant that is suitable for implantation into themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT Internationalapplication no. PCT/US/03307 (Publication No. WO 95/24929, entitled“Polymeric Gene Delivery System”, which claims priority to U.S. PatentApplication Serial No. 213,668, filed Mar. 15, 1994). PCT/US/03307describes a biocompatible, preferably biodegradable polymeric matrix forcontaining an exogenous gene under the control of an appropriatepromoter. The polymeric matrix is used to achieve sustained release ofthe exogenous gene in the patient. In accordance with the instantinvention, the nucleic acids described herein are encapsulated ordispersed within the biocompatible, preferably biodegradable polymericmatrix disclosed in PCT/US/03307. The polymeric matrix preferably is inthe form of a micro particle such as a micro sphere (wherein a nucleicacid is dispersed throughout a solid polymeric matrix) or a microcapsule(wherein a nucleic acid is stored in the core of a polymeric shell).Other forms of the polymeric matrix for containing the nucleic acids ofthe invention include films, coatings, gels, implants, and stents. Thesize and composition of the polymeric matrix device is selected toresult in favorable release kinetics in the tissue into which the matrixdevice is implanted. The size of the polymeric matrix device further isselected according to the method of delivery which is to be used,typically injection into a tissue or administration of a suspension byaerosol into the nasal and/or pulmonary areas. The polymeric matrixcomposition can be selected to have both favorable degradation rates andalso to be formed of a material which is bioadhesive, to furtherincrease the effectiveness of transfer when the device is administeredto a vascular surface. The matrix composition also can be selected notto degrade, but rather, to release by diffusion over an extended periodof time.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the nucleic acids of the invention to the subject.Biodegradable matrices are preferred. Such polymers may be natural orsynthetic polymers. Synthetic polymers are preferred. The polymer isselected based on the period of time over which release is desired,generally in the order of a few hours to a year or longer. Typically,release over a period ranging from between a few hours and three totwelve months is most desirable. The polymer optionally is in the formof a hydrogel that can absorb up to about 90% of its weight in water andfurther, optionally is cross-linked with multi-valent ions or otherpolymers.

In general, the nucleic acids of the invention are delivered using thebioerodible implant by way of diffusion, or more preferably, bydegradation of the polymeric matrix. Exemplary synthetic polymers whichcan be used to form the biodegradable delivery system include:polyamides, polycarbonates, polyalkylenes, polyalkylene glycols,polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols,polyvinyl ethers, polyvinyl esters, polyvinyl halides, polyglycolides,polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose,hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitrocelluloses, polymers of acrylic and methacrylic esters, methylcellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propylmethyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate,cellulose propionate, cellulose acetate butyrate, cellulose acetatephthalate, carboxylethyl cellulose, cellulose triacetate, cellulosesulphate sodium salt, poly(methylmethacrylate), poly(ethylmethacrylate),poly(butylmethacrylate), poly(isobutylmethacrylate),poly(hexylmethacrylate), poly(isodecylmethacrylate),poly(laurylmethacrylate), poly(phenylmethacrylate),poly(methylacrylate), poly(isopropylacrylate), poly(isobutylacrylate),poly(octadecylacrylate), polyethylene, polypropylene,poly(ethyleneglycol), poly(ethyleneoxide), poly(ethyleneterephthalate),poly(vinyl alcohols), polyvinyl acetate, poly vinyl chloride,polystyrene and polyvinylpyrrolidone.

Examples of non-biodegradable polymers include ethylene vinyl acetate,poly(meth)acrylic acid, polyamides, copolymers and mixtures thereof.

Examples of biodegradable polymers include synthetic polymers such aspolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid),and poly(lactide-cocaprolactone), and natural polymers such as alginateand other polysaccharides including dextran and cellulose, collagen,chemical derivatives thereof (substitutions, additions of chemicalgroups, for example, alkyl, alkylene, hydroxylations, oxidations, andother modifications routinely made by those skilled in the art), albuminand other hydrophilic proteins, zein and other prolamines andhydrophobic proteins, copolymers and mixtures thereof. In general, thesematerials degrade either by enzymatic hydrolysis or exposure to water invivo, by surface or bulk erosion.

Bioadhesive polymers of particular interest include bioerodiblehydrogels described by H. S. Sawhney, C. P. Pathak and J. A. Hubell inMacromolecules, 1993, 26, 581-587, the teachings of which areincorporated herein, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate). Thus, the invention provides acomposition of the above-described molecules of the invention for use asa medicament, methods for preparing the medicament and methods for thesustained release of the medicament in vivo.

Compaction agents also can be used in combination with a vector of theinvention. A “compaction agent”, as used herein, refers to an agent,such as a histone, that neutralizes the negative charges on the nucleicacid and thereby permits compaction of the nucleic acid into a finegranule. Compaction of the nucleic acid facilitates the uptake of thenucleic acid by the target cell. The compaction agents can be usedalone, e.g., to deliver an isolated nucleic acid of the invention in aform that is more efficiently taken up by the cell or, more preferably,in combination with one or more of the above-described vectors.

Other exemplary compositions that can be used to facilitate uptake by atarget cell of the nucleic acids of the invention include calciumphosphate and other chemical mediators of intracellular transport,microinjection compositions, electroporation and homologousrecombination compositions (e.g., for integrating a nucleic acid into apreselected location within the target cell chromosome).

The invention also provides methods for the diagnosis and therapy ofvascular and cardiovascular disorders. Such disorders include myocardialinfarction, stroke, arteriosclerosis, heart failure, and cardiachypertrophy.

The methods of the invention are useful in both the acute and theprophylactic treatment of any of the foregoing conditions. As usedherein, an acute treatment refers to the treatment of subjects having aparticular condition. Prophylactic treatment refers to the treatment ofsubjects at risk of having the condition, but not presently having orexperiencing is the symptoms of the condition.

In its broadest sense, the terms “treatment” or “to treat” refer to bothacute and prophylactic treatments. If the subject in need of treatmentis experiencing a condition (or has or is having a particularcondition), then treating the condition refers to ameliorating, reducingor eliminating the condition or one or more symptoms arising from thecondition. In some preferred embodiments, treating the condition refersto ameliorating, reducing or eliminating a specific symptom or aspecific subset of symptoms associated with the condition. If thesubject in need of treatment is one who is at risk of having acondition, then treating the subject refers to reducing the risk of thesubject having the condition.

Stroke (also referred to herein as ischemic stroke and/orcerebrovascular ischemia) is often cited as the third most common causeof death in the industrial world, ranking behind ischemic heart diseaseand cancer. Strokes are responsible for about 300,000 deaths annually inthe United States and are a leading cause of hospital admissions andlong-term disabilities. Accordingly, the socioeconomic impact of strokeand its attendant burden on society is practically immeasurable.

“Stroke” is defined by the World Health Organization as a rapidlydeveloping clinical sign of focal or global disturbance of cerebralfunction with symptoms lasting at least 24 hours. Strokes are alsoimplicated in deaths where there is no apparent cause other than aneffect of vascular origin.

Strokes are typically caused by blockages or occlusions of the bloodvessels to the brain or within the brain. With complete occlusion,arrest of cerebral circulation causes cessation of neuronal electricalactivity within seconds. Within a few minutes after the deterioration ofthe energy state and ion homeostasis, depletion of high energyphosphates, membrane ion pump failure, efflux of cellular potassium,influx of sodium chloride and water, and membrane depolarization occur.If the occlusion persists for more than five to ten minutes,irreversible damage results. With incomplete ischemia, however, theoutcome is difficult to evaluate and depends largely on residualperfusion and the availability of oxygen. After a thrombotic occlusionof a cerebral vessel, ischemia is rarely total. Some residual perfusionusually persists in the ischemic area, depending on collateral bloodflow and local perfusion pressure.

Cerebral blood flow can compensate for drops in mean arterial bloodpressure from 90 to 60 mm Hg by autoregulation. This phenomenon involvesdilatation of downstream resistant vessels. Below the lower level ofautoregulation (about 60 mm Hg), vasodilatation is inadequate and thecerebral blood flow falls. The brain, however, has perfusion reservesthat can compensate for the fall in cerebral blood flow. This reserveexists because under normal conditions only about 35% of the oxygendelivered by the blood is extracted. Therefore, increased oxygenextraction can take place, provided that normoxia and normocapnea exist.When distal blood pressure falls below approximately 30 mm Hg, the twocompensatory mechanisms (autoregulation and perfusion reserve) areinadequate to prevent failure of oxygen delivery.

As blood flow drops below the ischemic threshold of 23 ml/100 g/minute,symptoms of tissue hypoxia develop. Severe ischemia may be lethal. Whenthe ischemia is moderate, it will result in “penumbra.” In theneurological context, penumbra refers to a zone of brain tissue withmoderate ischemia and paralyzed neuronal function, which is reversiblewith restoration of adequate perfusion. The penumbra forms a zone ofcollaterally perfused tissue surrounding a core of severe ischemia inwhich an infarct has developed. In other words, the penumbra is thetissue area that can be saved, and is essentially in a state betweenlife and death.

Although an ischemic event can occur anywhere in the vascular system,the carotid artery bifurcation and the origin of the internal carotidartery are the most frequent sites for thrombotic occlusions of cerebralblood vessels, which result in cerebral ischemia. The symptoms ofreduced blood flow due to stenosis or thrombosis are similar to thosecaused by middle cerebral artery disease. Flow through the ophthalmicartery is often affected sufficiently to produce amaurosis fugax ortransient monocular blindness. Severe bilateral internal carotid arterystenosis may result in cerebral hemispheric hypoperfusion. Thismanifests with acute headache ipsilateral to the acutely ischemichemisphere. Occlusions or decrease of the blood flow with resultingischemia of one anterior cerebral artery distal to the anteriorcommunicating artery produces motor and cortical sensory symptoms in thecontralateral leg and, less often, proximal arm. Other manifestations ofocclusions or underperfusion of the anterior cerebral artery includegait ataxia and sometimes urinary incontinence due to damage to theparasagital frontal lobe. Language disturbances manifested as decreasedspontaneous speech may accompany generalized depression of psychomotoractivity.

Most ischemic strokes involve portions or all of the territory of themiddle cerebral artery with emboli from the heart or extracranialcarotid arteries accounting for most cases. Emboli may occlude the mainstem of the middle cerebral artery, but more frequently produce distalocclusion of either the superior or the inferior branch. Occlusions ofthe superior branch cause weakness and sensory loss that are greatest inthe face and arm. Occlusions of the posterior cerebral artery distal toits penetrating branches cause complete contralateral loss of vision.Difficulty in reading (dyslexia) and in performing calculations(dyscalculia) may follow ischemia of the dominant posterior cerebralartery. Proximal occlusion of the posterior cerebral artery causesischemia of the branches penetrating to calamic and limbic structures.The clinical results are hemisensory disturbances that may chronicallychange to intractable pain of the defective side (thalamic pain).

A subject having a stroke is so diagnosed by symptoms experienced and/orby a physical examination including interventional andnon-interventional diagnostic tools such as CT and MR imaging. Themethods of the invention are advantageous for the treatment of variousclinical presentations of stroke subjects. A subject having a stroke maypresent with one or more of the following symptoms: paralysis, weakness,decreased sensation and/or vision, numbness, tingling, aphasia (e.g.,inability to speak or slurred speech, difficulty reading or writing),agnosia (i.e., inability to recognize or identify sensory stimuli), lossof memory, co-ordination difficulties, lethargy, sleepiness orunconsciousness, lack of bladder or bowel control and cognitive decline(e.g., dementia, limited attention span, inability to concentrate).Using medical imaging techniques, it may be possible to identify asubject having a stroke as one having an infarct or one havinghemorrhage in the brain.

An important embodiment of the invention is treatment of a subject withan abnormally elevated risk of an ischemic stroke. As used herein,subjects having an abnormally elevated risk of an ischemic stroke are acategory determined according to conventional medical practice (seeearlier discussion); such subjects may also be identified inconventional medical practice as having known risk factors for stroke orhaving increased risk of cerebrovascular events. This category includes,for example, subjects which are having elected vascular surgery.Typically, the risk factors associated with cardiac disease are the sameas are associated with stroke. The primary risk factors includehypertension, hypercholesterolemia, and smoking. Atrial fibrillation orrecent myocardial infarction are also important risk factors. Inaddition, modified levels of expression of a nucleic acid moleculeselected from the group consisting of Fit-1, vacuolar ATPase, CD44,Lot-1, AA892598, and Mrg-1, or an expression product thereof, are also,according to the present invention, important risk factors.

As used herein, subjects having an abnormally elevated risk of anischemic stroke also include individuals undergoing surgical ordiagnostic procedures which risk release of emboli, lowering of bloodpressure or decrease in blood flow to the brain, such as carotidendarterectomy, brain angiography, neurosurgical procedures in whichblood vessels are compressed or occluded, cardiac catheterization,angioplasty, including balloon angioplasty, coronary by-pass surgery, orsimilar procedures. Subjects having an abnormally elevated risk of anischemic stroke also include individuals having any cardiac conditionthat may lead to decreased blood flow to the brain, such as atrialfibrillation, ventrical tachycardia, dilated cardiomyopathy and othercardiac conditions requiring anticoagulation. Subjects having anabnormally elevated risk of an ischemic stroke also include individualshaving conditions including arteriopathy or brain vasculitis, such asthat caused by lupus, congenital diseases of blood vessels, such asCADASIL syndrome, or migraine, especially prolonged episodes.

The treatment of stroke can be for patients who have experienced astroke or can be a prophylactic treatment. Short term prophylactictreatment is indicated for subjects having surgical or diagnosticprocedures which risk release of emboli, lowering of blood pressure ordecrease in blood flow to the brain, to reduce the injury due to anyischemic event that occurs as a consequence of the procedure. Longerterm or chronic prophylactic treatment is indicated for subjects havingcardiac conditions that may lead to decreased blood flow to the brain,or conditions directly affecting brain vasculature. If prophylactic,then the treatment is for subjects having an abnormally elevated risk ofan ischemic stroke, as described above. If the subject has experienced astroke, then the treatment can include acute treatment. Acute treatmentfor stroke subjects means administration of an agent of the invention atthe onset of symptoms of the condition or within 48 hours of the onset,preferably within 24 hours, more preferably within 12 hours, morepreferably within 6 hours, and even more preferably within 3 hours ofthe onset of symptoms of the condition.

Criteria for defining hypercholesterolemic and/or hypertriglyceridemicsubjects are well known in the art (see, e.g., “Harrison's”).Hypercholesterolemic subjects and hypertriglyceridemic subjects areassociated with increased incidence of premature coronary heart disease.A hypercholesterolemic subject has an LDL level of >160 mg/dL or >130mg/dL and at least two risk factors selected from the group consistingof male gender, family history of premature coronary heart disease,cigarette smoking (more than 10 per day), hypertension, low HDL (<35mg/dL), diabetes mellitus, hyperinsulinemia, abdominal obesity, highlipoprotein (a), and personal history of cerebrovascular disease orocclusive peripheral vascular disease. A hypertriglyceridemic subjecthas a triglyceride (TG) level of >250 mg/dL. Thus, a hyperlipidemicsubject is defined as one whose cholesterol and triglyceride levelsequal or exceed the limits set as described above for both thehypercholesterolemic and hypertriglyceridemic subjects.

“Myocardial infarction” is a focus of necrosis resulting from inadequateperfusion of the cardiac tissue. Myocardial infarction generally occurswith the abrupt decrease in coronary blood flow that follows athrombotic occlusion of a coronary artery previously narrowed byatherosclerosis. Generally, infarction occurs when an atheroscleroticplaque fissures, ruptures, or ulcerates, and a mural thrombus formsleading to coronary artery occlusion.

The diagnosis of myocardial infarction in a subject determines the needfor treating the subject according to the methods of the invention. Anumber of laboratory tests, well known in the art, are described, forexample, in Harrison's. Generally, the tests may be divided into fourmain categories: (1) nonspecific indexes of tissue necrosis andinflammation, (2) electrocardiograms, (3) serum enzyme changes (e.g.,creatine phosphokinase levels), and (4) cardiac imaging. A person ofordinary skill in the art could easily apply any of the foregoing teststo determine when a subject is at risk, is suffering, or has suffered, amyocardial infarction. In addition, decreased levels of expression of anucleic acid molecule selected from the group consisting of Fit-1,vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1, or an expressionproduct thereof, are also, according to the present invention, importantrisk factors. A positively identified subject would thus benefit from amethod of treatment of the invention.

According to the invention, the method involves administering to asubject having a myocardial infarction any of the foregoing molecules(Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1) in an amounteffective to treat the cardiovascular disorder in the subject. By“having a myocardial infarction” it is meant that the subject is at riskof developing, is currently having, or has suffered a myocardialinfarction. It is believed that immediate administration of the moleculewould greatly benefit the subject by inhibiting apoptotic cell-death ofcardiomyocytes (the cells mostly affected by the infarct) prior to, orfollowing the infarct. By “immediate” it is meant that administrationoccurs before (if it is diagnosed in time), or within 48 hours from themyocardial infarct, although administration up to 14 days after theepisode may also be beneficial to the subject.

Another important embodiment of the invention is the treatment ofischemic injury resulting from arteriosclerosis. Arteriosclerosis is aterm used to describe a thickening and hardening of the arterial wall.It is believed to be responsible for the majority of deaths in theUnited States and in most westernized societies. Atherosclerosis is onetype of arteriosclerosis that is believed to be the cause of mostcoronary artery disease, aortic aneurysm and arterial disease of thelower extremities (including peripheral vascular arteriopathy), as wellas contributing to cerebrovascular disease. Atherosclerosis is theleading cause of death in the United States.

A normal artery typically is lined on its inner-side only by a singlelayer of endothelial cells, the intima. The intima overlays the media,which contains only a single cell type, the smooth muscle cell. Theouter-most layer of the artery is the adventitia. With aging, there is acontinuous increase in the thickness of the intima, believed to resultin part from migration and proliferation of smooth muscle cells from themedia. A similar increase in the thickness of the intima also occurs asa result of various traumatic events or interventions, such as occurswhen, for example, a balloon dilatation procedure causes injury to thevessel wall. The invention is used in connection with treating ischemicinjury resulting from arteriosclerotic conditions. An arterioscleroticcondition as used herein means classical atherosclerosis, acceleratedatherosclerosis, atherosclerosis lesions and any other arterioscleroticconditions characterized by undesirable endothelial and/or vascularsmooth muscle cell proliferation, including vascular complications ofdiabetes.

Another important embodiment of the invention is the treatment of heartfailure. Heart failure is a clinical syndrome of diverse etiologieslinked by the common denominator of impaired heart pumping and ischaracterized by the failure of the heart to pump blood commensuratewith the requirements of the metabolizing tissues, or to do so only froman elevating filling pressure.

Another important embodiment of the invention is the treatment ofcardiac hypertrophy. This condition is typically characterized by leftventricular hypertrophy, usually of a nondilated chamber, withoutobvious antecedent cause. Current methods of diagnosis include theelectrocardiogram and the echocardiogram. Many patients, however, areasymptomatic and may be relatives of patients with known disease.Unfortunately, the first manifestation of the disease may be suddendeath, frequently occurring in children and young adults, often duringor after physical exertion.

Agents for reducing the risk of or treating a cardiovascular disorderinclude those selected from the group consisting of anti-inflammatoryagents, anti-thrombotic agents, anti-platelet agents, fibrinolyticagents, lipid reducing agents, direct thrombin inhibitors, glycoproteinIIb/IIIa receptor inhibitors, agents that bind to cellular adhesionmolecules and inhibit the ability of white blood cells to attach to suchmolecules (e.g. anti-cellular adhesion molecule antibodies), calciumchannel blockers, beta-adrenergic receptor blockers, cyclooxygenase-2inhibitors, angiotensin system inhibitors, and/or any combinationsthereof. One preferred agent is aspirin.

The mode of administration and dosage of a therapeutic agent of theinvention will vary with the particular stage of the condition beingtreated, the age and physical condition of the subject being treated,the duration of the treatment, the nature of the concurrent therapy (ifany), the specific route of administration, and the like factors withinthe knowledge and expertise of the health practitioner.

As described herein, the agents of the invention are administered ineffective amounts to treat any of the foregoing cardiovasculardisorders. In general, an effective amount is any amount that can causea beneficial change in a desired tissue of a subject. Preferably, aneffective amount is that amount sufficient to cause a favorablephenotypic change in a particular condition such as a lessening,alleviation or elimination of a symptom or of a condition as a whole.

In general, an effective amount is that amount of a pharmaceuticalpreparation that alone, or together with further doses, produces thedesired response. This may involve only slowing the progression of thecondition temporarily, although more preferably, it involves halting theprogression of the condition permanently or delaying the onset of orpreventing the condition from occurring. This can be monitored byroutine methods. Generally, doses of active compounds would be fromabout 0.01 mg/kg per day to 1000 mg/kg per day. It is expected thatdoses ranging from 50-500 mg/kg will be suitable, preferably orally andin one or several administrations per day.

Such amounts will depend, of course, on the particular condition beingtreated, the severity of the condition, the individual patientparameters including age, physical condition, size and weight, theduration of the treatment, the nature of concurrent therapy (if any),the specific route of administration and like factors within theknowledge and expertise of the health practitioner. Lower doses willresult from certain forms of administration, such as intravenousadministration. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits. Multiple doses isper day are contemplated to achieve appropriate systemic levels ofcompounds. It is preferred generally that a maximum dose be used, thatis, the highest safe dose according to sound medical judgment. It willbe understood by those of ordinary skill in the art, however, that apatient may insist upon a lower dose or tolerable dose for medicalreasons, psychological reasons or for virtually any other reasons.

The agents of the invention may be combined, optionally, with apharmaceutically-acceptable carrier to form a pharmaceuticalpreparation. The term “pharmaceutically-acceptable carrier,” as usedherein, means one or more compatible solid or liquid fillers, diluentsor encapsulating substances which are suitable for administration into ahuman. The term “carrier” denotes an organic or inorganic ingredient,natural or synthetic, with which the active ingredient is combined tofacilitate the application. The components of the pharmaceuticalcompositions also are capable of being co-mingled with the molecules ofthe present invention, and with each other, in a manner such that thereis no interaction which would substantially impair the desiredpharmaceutical efficacy. In some aspects, the pharmaceuticalpreparations comprise an agent of the invention in an amount effectiveto treat a disorder.

The pharmaceutical preparations may contain suitable buffering agents,including: acetic acid in a salt; citric acid in a salt; boric acid in asalt; or phosphoric acid in a salt. The pharmaceutical compositions alsomay contain, optionally, suitable preservatives, such as: benzalkoniumchloride; chlorobutanol; parabens or thimerosal.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular drug selected, theseverity of the condition being treated and the dosage required fortherapeutic efficacy. The methods of the invention, generally speaking,may be practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels of theactive compounds without causing clinically unacceptable adverseeffects. Such modes of administration include oral, rectal, topical,nasal, intradermal, transdermal, or parenteral routes. The term“parenteral” includes subcutaneous, intravenous, intramuscular, orinfusion. Intravenous or intramuscular routes are not particularlysuitable for long-term therapy and prophylaxis. As an example,pharmaceutical compositions for the acute treatment of subjects having amigraine headache may be formulated in a variety of different ways andfor a variety of administration modes including tablets, capsules,powders, suppositories, injections and nasal sprays.

The pharmaceutical preparations may conveniently be presented in unitdosage form and may be prepared by any of the methods well-known in theart of pharmacy. All methods include the step of bringing the activeagent into association with a carrier which constitutes one or moreaccessory ingredients. In general, the compositions are prepared byuniformly and intimately bringing the active compound into associationwith a liquid carrier, a finely divided solid carrier, or both, andthen, if necessary, shaping the product.

Compositions suitable for oral administration may be presented asdiscrete units, such as capsules, tablets, lozenges, each containing apredetermined amount of the active compound. Other compositions includesuspensions in aqueous liquids or non-aqueous liquids such as a syrup,elixir or an emulsion.

Compositions suitable for parenteral administration convenientlycomprise a sterile aqueous preparation of an agent of the invention,which is preferably isotonic with the blood of the recipient. Thisaqueous preparation may be formulated according to known methods usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation also may be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono ordi-glycerides. In addition, fatty acids such as oleic acid may be usedin the preparation of injectables. Formulations suitable for oral,subcutaneous, intravenous, intramuscular, etc. administrations can befound in Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.

The term “permit entry” of a molecule into a cell according to theinvention has the following meanings depending upon the nature of themolecule. For an isolated nucleic acid it is meant to describe entry ofthe nucleic acid through the cell membrane and into the cell nucleus,where upon the “nucleic acid transgene” can utilize the cell machineryto produce functional polypeptides encoded by the nucleic acid. By“nucleic acid transgene” it is meant to describe all of the nucleicacids of the invention with or without the associated vectors. For apolypeptide, it is meant to describe entry of the polypeptide throughthe cell membrane and into the cell cytoplasm, and if necessary,utilization of the cell cytoplasmic machinery to functionally modify thepolypeptide (e.g., to an active form).

Various techniques may be employed for introducing nucleic acids of theinvention into cells, depending on whether the nucleic acids areintroduced in vitro or in vivo in a host. Such techniques includetransfection of nucleic acid-CaPO₄ precipitates, transfection of nucleicacids associated with DEAE, transfection with a retrovirus including thenucleic acid of interest, liposome mediated transfection, and the like.For certain uses, it is preferred to target the nucleic acid toparticular cells. In such instances, a vehicle used for delivering anucleic acid of the invention into a cell (e.g., a liposome, aretrovirus, or other virus) can have a targeting molecule attachedthereto. For example, a molecule such as an antibody specific for asurface membrane protein on the target cell or a ligand for a receptoron the target cell can be bound to or incorporated within the nucleicacid delivery vehicle. For example, where liposomes are employed todeliver the nucleic acids of the invention, proteins which bind to asurface membrane protein associated with endocytosis may be incorporatedinto the liposome formulation for targeting and/or to facilitate uptake.Such proteins include capsid proteins or fragments thereof tropic for aparticular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half life, and the like.Polymeric delivery systems also have been used successfully to delivernucleic acids into cells, as is known by those skilled in the art. Suchsystems even permit oral delivery of nucleic acids.

Other delivery systems can include time release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of an agent of the present invention, increasingconvenience to the subject and the physician. Many types of releasedelivery systems are available and known to those of ordinary skill inthe art. They include polymer base systems such aspoly(lactide-glycolide), copolyoxalates, polycaprolactones,polyesteramides, polyorthoesters, polyhydroxybutyric acid, andpolyanhydrides. Microcapsules of the foregoing polymers containing drugsare described in, for example, U.S. Pat. No. 5,075,109. Delivery systemsalso include non-polymer systems that are: lipids including sterols suchas cholesterol, cholesterol esters and fatty acids or neutral fats suchas mono-, di-, and tri-glycerides; hydrogel release systems; sylasticsystems; peptide based systems; wax coatings; compressed tablets usingconventional binders and excipients; partially fused implants; and thelike. Specific examples include, but are not limited to: (a) erosionalsystems in which an agent of the invention is contained in a form withina matrix such as those described in U.S. Pat. Nos. 4,452,775; 4,675,189;and 5,736,152; and (b) diffusional systems in which an active componentpermeates at a controlled rate from a polymer such as described in U.S.Pat. Nos. 3,854,480; 5,133,974: and 5,407,686. In addition, pump-basedhardware delivery systems can be used, some of which are adapted forimplantation.

Use of a long-term sustained release implant may be desirable. Long-termrelease, as used herein, means that the implant is constructed andarranged to deliver therapeutic levels of the active ingredient for atleast 30 days, and preferably 60 days. Long-term sustained releaseimplants are well-known to those of ordinary skill in the art andinclude some of the release systems described above. Specific examplesinclude, but are not limited to, long-term sustained release implantsdescribed in U.S. Pat. No. 4,748,024, and Canadian Patent No. 1330939.

The invention also involves the administration, and in some embodimentsco-administration, of agents other than the molecules of the invention(Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1, nucleic acidsand polypeptides, and/or fragments thereof) that when administered ineffective amounts can act cooperatively, additively or synergisticallywith a molecule of the invention to: (i) modulate cardiac cellanti-apoptotic activity, and (ii) treat any of the conditions in whichcardiac cell anti-apoptotic activity of a molecule of the invention isinvolved. Agents other than the molecules of the invention includeanti-inflammatory agents, anti-thrombotic agents, anti-coagulants,anti-platelet agents, fibrinolytic agents, lipid reducing agents, directthrombin inhibitors, glycoprotein IIb/IIIa receptor inhibitors, agentsthat bind to cellular adhesion molecules and inhibit the ability ofwhite blood cells to attach to such molecules, calcium channel blockers,beta-adrenergic receptor blockers, cyclooxygenase-2 inhibitors,angiotensin system inhibitors, anti-hypertensive agents, and/orcombinations thereof.

“Anti-inflammatory” agents include Alclofenac; AlclometasoneDipropionate; Algestone Acetonide; Alpha Amylase; Amcinafal; Amcinafide;Amfenac Sodium; Amiprilose Hydrochloride; Anakinra; Anirolac;Anitrazafen; Apazone; Balsalazide Disodium; Bendazac; Benoxaprofen;Benzydamine Hydrochloride; Bromelains; Broperamole; Budesonide;Carprofen; Cicloprofen; Cintazone; Cliprofen; Clobetasol Propionate;Clobetasone Butyrate; Clopirac; Cloticasone Propionate; CormethasoneAcetate; Cortodoxone; Deflazacort; Desonide; Desoximetasone;Dexamethasone Dipropionate; Diclofenac Potassium; Diclofenac Sodium;Diflorasone Diacetate; Diflumidone Sodium; Diflunisal; Difluprednate;Diftalone; Dimethyl Sulfoxide; Drocinonide; Endrysone; Enlimomab;Enolicam Sodium; Epirizole; Etodolac; Etofenamate; Felbinac; Fenamole;Fenbufen; Fenclofenac; Fenclorac; Fendosal; Fenpipalone; Fentiazac;Flazalone; Fluazacort; Flufenamic Acid; Flumizole; Flunisolide Acetate;Flunixin; Flunixin Meglumine; Fluocortin Butyl; Fluorometholone Acetate;Fluquazone; Flurbiprofen; Fluretofen; Fluticasone Propionate;Furaprofen; Furobufen; Halcinonide; Halobetasol Propionate; HalopredoneAcetate; Ibufenac; Ibuprofen; Ibuprofen Aluminum; Ibuprofen Piconol;Ilonidap; Indomethacin; Indomethacin Sodium; Indoprofen; Indoxole;Intrazole; Isoflupredone Acetate; Isoxepac; Isoxicam; Ketoprofen;Lofemizole Hydrochloride; Lornoxicam; Loteprednol Etabonate;Meclofenamate Sodium; Meclofenamic Acid; Meclorisone Dibutyrate;Mefenamic Acid; Mesalamine; Meseclazone; Methylprednisolone Suleptanate;Morniflumate; Nabumetone; Naproxen; Naproxen Sodium; Naproxol; Nimazone;Olsalazine Sodium; Orgotein; Orpanoxin; Oxaprozin; Oxyphenbutazone;Paranyline Hydrochloride; Pentosan Polysulfate Sodium; PhenbutazoneSodium Glycerate; Pirfenidone; Piroxicam; Piroxicam Cinnamate; PiroxicamOlamine; Pirprofen; Prednazate; Prifelone; Prodolic Acid; Proquazone;Proxazole; Proxazole Citrate; Rimexolone; Romazarit; Salcolex;Salnacedin; Salsalate; Salycilates; Sanguinarium Chloride; Seclazone;Sermetacin; Sudoxicam; Sulindac; Suprofen; Talmetacin; Talniflumate;Talosalate; Tebufelone; Tenidap; Tenidap Sodium; Tenoxicam; Tesicam;Tesimide; Tetrydamine; Tiopinac; Tixocortol Pivalate; Tolmetin; TolmetinSodium; Triclonide; Triflumidate; Zidometacin; Glucocorticoids; andZomepirac Sodium. One preferred anti-inflammatory agent is aspirin.

“Anti-thrombotic” and/or “fibrinolytic” agents include plasminogen (toplasmin via interactions of prekallikrein, kininogens, Factors XII,XIIIa, plasminogen proactivator, and tissue plasminogen activator[TPA])Streptokinase; Urokinase: Anisoylated Plasminogen-StreptokinaseActivator Complex; Pro-Urokinase; (Pro-UK); rTPA (alteplase or activase;“r” denotes recombinant); rPro-UK; Abbokinase; Eminase; SreptaseAnagrelide Hydrochloride; Bivalirudin; Dalteparin Sodium; DanaparoidSodium; Dazoxiben Hydrochloride; Efegatran Sulfate; Enoxaparin Sodium;Ifetroban; Ifetroban Sodium; Tinzaparin Sodium; Retaplase; Trifenagrel;Warfarin; and Dextrans.

“Anti-platelet” agents include Clopridogrel; Sulfinpyrazone; Aspirin;Dipyridamole; Clofibrate; Pyridinol Carbamate; PGE; Glucagon;Antiserotonin drugs; Caffeine; Theophyllin Pentoxifyllin; Ticlopidine;and Anagrelide.

“Lipid reducing” agents include gemfibrozil, cholystyramine, colestipol,nicotinic acid, probucol lovastatin, fluvastatin, simvastatin,atorvastatin, pravastatin, and cirivastatin.

“Direct thrombin inhibitors” include hirudin, hirugen, hirulog,agatroban, PPACK, and thrombin aptamers.

“Glycoprotein IIb/IIIa receptor inhibitors” embraces both antibodies andnon-antibodies, and include, but are not limited, to ReoPro (abcixamab),lamifiban, and tirofiban.

“Calcium channel blockers” are a chemically diverse class of compoundshaving important therapeutic value in the control of a variety ofdiseases including several cardiovascular disorders, such ashypertension, angina, and cardiac arrhythmias (Fleckenstein, Cir. Res.v. 52, (suppl. 1), p. 13-16 (1983); Fleckenstein, Experimental Facts andTherapeutic Prospects, John Wiley, New York (1983); McCall, D., CurrPract Cardiol, v. 10, p. 1-11 (1985)). Calcium channel blockers are aheterogeneous group of drugs that prevent or slow the entry of calciuminto cells by regulating cellular calcium channels. (Remington, TheScience and Practice of Pharmacy, Nineteenth Edition, Mack PublishingCompany, Eaton, Pa., p. 963 (1995)). Most of the currently availablecalcium channel blockers, and useful according to the present invention,belong to one of three major chemical groups of drugs, thedihydropyridines, such as nifedipine, the phenyl alkyl amines, such asverapamil, and the benzothiazepines, such as diltiazem. Other calciumchannel blockers useful according to the invention, include, but are notlimited to, aminone, amlodipine, bencyclane, felodipine, fendiline,flunarizine, isradipine, nicardipine, nimodipine, perhexylene,gallopamil, tiapamil and tiapamil analogues (such as 1993RO-11-2933),phenyloin, barbiturates, and the peptides dynorphin, omega-conotoxin,and omega-agatoxin, and the like and/or pharmaceutically acceptablesalts thereof.

“Beta-adrenergic receptor blocking agents” are a class of drugs thatantagonize the cardiovascular effects of catecholamines in anginapectoris, hypertension, and cardiac arrhythmias. Beta-adrenergicreceptor blockers include, but are not limited to, atenolol, acebutolol,alprenolol, befunolol, betaxolol, bunitrolol, carteolol, celiprolol,hydroxalol, indenolol, labetalol, levobunolol, mepindolol, methypranol,metindol, metoprolol, metrizoranolol, oxprenolol, pindolol, propranolol,practolol, practolol, sotalolnadolol, tiprenolol, tomalolol, timolol,bupranolol, penbutolol, trimepranol,2-(3-(1,1-dimethylethyl)-amino-2-hydroxypropoxy)-3-pyridenecarbonitrilHCl,1-butylamino-3-(2,5-dichlorophenoxy)-2-propanol,1-isopropylamino-3-(4-(2-cyclopropylmethoxyethyl)phenoxy)-2-propanol,3-isopropylamino-1-(7-methylindan-4-yloxy)-2-butanol,2-(3-t-butylamino-2-hydroxy-propylthio)-4-(5-carbamoyl-2-thienyl)thiazol,7-(2-hydroxy-3-t-butylaminpropoxy)phthalide.The above-identified compounds can be used as isomeric mixtures, or intheir respective levorotating or dextrorotating form.

Cyclooxygenase-2 (COX-2) is a recently identified form of acyclooxygenase. “Cyclooxygenase” is an enzyme complex present in mosttissues that produces various prostaglandins and thromboxanes fromarachidonic acid. Non-steroidal, anti-inflammatory drugs exert most oftheir anti-inflammatory, analgesic and antipyretic activity and inhibithormone-induced uterine contractions and certain types of cancer growththrough inhibition of the cyclooxygenase (also known as prostaglandinG/H synthase and/or prostaglandin-endoperoxide synthase). Initially,only one form of cyclooxygenase was known, the “constitutive enzyme” orcyclooxygenase-1 (COX-1). It and was originally identified in bovineseminal vesicles.

Cyclooxygenase-2 (COX-2) has been cloned, sequenced and characterizedinitially from chicken, murine and human sources (see, e.g., U.S. Pat.No. 5,543,297, issued Aug. 6, 1996 to Cromlish et al., and assigned toMerck Frosst Canada, Inc., Kirkland, Calif., entitled: “Humancyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity”). This enzyme is distinct from COX-1. COX-2 is rapidly andreadily inducible by a number of agents including mitogens, endotoxin,hormones, cytokines and growth factors. As prostaglandins have bothphysiological and pathological roles, the constitutive enzyme, COX-1, isresponsible, in large part, for endogenous basal release ofprostaglandins and hence is important in their physiological functionssuch as the maintenance of gastrointestinal integrity and renal bloodflow. By contrast, it is believed that the inducible form, COX-2, ismainly responsible for the pathological effects of prostaglandins whererapid induction of the enzyme would occur in response to such agents asinflammatory agents, hormones, growth factors, and cytokines. Therefore,it is believed that a selective inhibitor of COX-2 has similaranti-inflammatory, antipyretic and analgesic properties to aconventional non-steroidal anti-inflammatory drug, and in additioninhibits hormone-induced uterine contractions and also has potentialanti-cancer effects, but with reduced side effects. In particular, suchCOX-2 inhibitors are believed to have a reduced potential forgastrointestinal toxicity, a reduced potential for renal side effects, areduced effect on bleeding times and possibly a decreased potential toinduce asthma attacks in aspirin-sensitive asthmatic subjects, and aretherefore useful according to the present invention.

A number of selective “COX-2 inhibitors” are known in the art. Theseinclude, but are not limited to, COX-2 inhibitors described in U.S. Pat.No. 5,474,995 “Phenyl heterocycles as COX-2 inhibitors”; U.S. Pat. No.5,521,213 “Diaryl bicyclic heterocycles as inhibitors ofcyclooxygenase-2”; U.S. Pat. No. 5,536,752 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,550,142 “Phenyl heterocycles as COX-2inhibitors”; U.S. Pat. No. 5,552,422 “Aryl substituted 5,5 fusedaromatic nitrogen compounds as anti-inflammatory agents”; U.S. No. Pat.5,604,253 “N-Benzylindol-3-yl propanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,604,260“5-Methanesulfonamido-1-indanones as an inhibitor of cyclooxygenase-2”;U.S. Pat. No. 5,639,780 N-Benzyl indol-3-yl butanoic acid derivatives ascyclooxygenase inhibitors”; U.S. Pat. No. 5,677,318Diphenyl-1,2-3-thiadiazoles as anti-inflammatory agents”; U.S. Pat. No.5,691,374 “Diaryl-5-oxygenated-2-(5H)-furanones as COX-2 inhibitors”;U.S. Pat. No. 5,698,584 “3,4-Diaryl-2-hydroxy-2,5-dihydrofurans asprodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,710,140 “Phenylheterocycles as COX-2 inhibitors”; U.S. Pat. No. 5,733,909 “Diphenylstilbenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No. 5,789,413“Alkylated styrenes as prodrugs to COX-2 inhibitors”; U.S. Pat. No.5,817,700 “Bisaryl cyclobutenes derivatives as cyclooxygenaseinhibitors”; U.S. Pat. No. 5,849,943 “Stilbene derivatives useful ascyclooxygenase-2 inhibitors”; U.S. Pat. No. 5,861,419 “Substitutedpyridines as selective cyclooxygenase-2 inhibitors”; U.S. Pat. No.5,922,742 “Pyridinyl-2-cyclopenten-1-ones as selective cyclooxygenase-2inhibitors”; U.S. Pat. No. 5,925,631 “Alkylated styrenes as prodrugs toCOX-2 inhibitors”; all of which are commonly assigned to Merck FrosstCanada, Inc. (Kirkland, Calif. or Merck & Co., Inc. (Rahway, N.J.).Additional COX-2 inhibitors are also described in U.S. Pat. No.5,643,933, assigned to G. D. Searle & Co. (Skokie, Ill.), entitled:“Substituted sulfonylphenylheterocycles as cyclooxygenase-2 and5-lipoxygenase inhibitors.”

A number of the above-identified COX-2 inhibitors are prodrugs ofselective COX-2 inhibitors, and exert their action by conversion in vivoto the active and selective COX-2 inhibitors. The active and selectiveCOX-2 inhibitors formed from the above-identified COX-2 inhibitorprodrugs are described in detail in WO 95/00501, published Jan. 5, 1995,WO 95/18799, published Jul. 13, 1995 and U.S. Pat. No. 5,474,995, issuedDec. 12, 1995. Given the teachings of U.S. Pat. No. 5,543,297, entitled:“Human cyclooxygenase-2 cDNA and assays for evaluating cyclooxygenase-2activity,” a person of ordinary skill in the art would be able todetermine whether an agent is a selective COX-2 inhibitor or a precursorof a COX-2 inhibitor, and therefore part of the present invention.

An “angiotensin system inhibitor” is an agent that interferes with thefunction, synthesis or catabolism of angiotensin II. These agentsinclude, but are not limited to, angiotensin-converting enzyme (ACE)inhibitors, angiotensin II antagonists, angiotensin II receptorantagonists, agents that activate the catabolism of angiotensin II, andagents that prevent the synthesis of angiotensin I from whichangiotensin II is ultimately derived. The renin-angiotensin system isinvolved in the regulation of hemodynamics and water and electrolytebalance. Factors that lower blood volume, renal perfusion pressure, orthe concentration of Na⁺ in plasma tend to activate the system, whilefactors that increase these parameters tend to suppress its function.

Angiotensin I and angiotensin II are synthesized by the enzymaticrenin-angiotensin pathway. The synthetic process is initiated when theenzyme renin acts on angiotensinogen, a pseudoglobulin in blood plasma,to produce the decapeptide angiotensin I. Angiotensin I is converted byangiotensin converting enzyme (ACE) to angiotensin II (angiotensin-[1-8]octapeptide). The latter is an active pressor substance which has beenimplicated as a causative agent in several forms of hypertension invarious mammalian species, e.g., humans.

Angiotensin (renin-angiotensin) system inhibitors are compounds that actto interfere with the production of angiotensin II from angiotensinogenor angiotensin I or interfere with the activity of angiotensin II. Suchinhibitors are well known to those of ordinary skill in the art andinclude compounds that act to inhibit the enzymes involved in theultimate production of angiotensin II, including renin and ACE. Theyalso include compounds that interfere with the activity of angiotensinII, once produced. Examples of classes of such compounds includeantibodies (e.g., to renin), amino acids and analogs thereof (includingthose conjugated to larger molecules), peptides (including peptideanalogs of angiotensin and angiotensin I), pro-renin related analogs,etc. Among the most potent and useful renin-angiotensin systeminhibitors are renin inhibitors, ACE inhibitors, and angiotensin IIantagonists. In a preferred embodiment of the invention, therenin-angiotensin system inhibitors are renin inhibitors, ACEinhibitors, and angiotensin II antagonists.

“Angiotensin II antagonists” are compounds which interfere with theactivity of angiotensin II by binding to angiotensin II receptors andinterfering with its activity. Angiotensin II antagonists are well knownand include peptide compounds and non-peptide compounds. Mostangiotensin II antagonists are slightly modified congeners in whichagonist activity is attenuated by replacement of phenylalanine inposition 8 with some other amino acid; stability can be enhanced byother replacements that slow degeneration in vivo. Examples ofangiotensin II antagonists include: peptidic compounds (e.g., saralasin,[(San¹)(Val⁵)(Ala⁸)]angiotensin-(1-8) octapeptide and related analogs);N-substituted imidazole-2-one (U.S. Pat. No. 5,087,634); imidazoleacetate derivatives including 2-N-butyl-4-chloro-1-(2-chlorobenzile),imidazole-5-acetic acid (see Long et al., J. Pharmacol. Exp. Ther.247(1), 1-7 (1988));4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid andanalog derivatives (U.S. Pat. No. 4,816,463); N2-tetrazolebeta-glucuronide analogs (U.S. Pat. No. 5,085,992); substitutedpyrroles, pyrazoles, and tryazoles (U.S. Pat. No. 5,081,127); phenol andheterocyclic derivatives such as 1,3-imidazoles (U.S. Pat. No.5,073,566); imidazo-fused 7-member ring heterocycles (U.S. Pat. No.5,064,825); peptides (e.g., U.S. Pat. No. 4,772,684); antibodies toangiotensin II (e.g., U.S. Pat. No. 4,302,386); and aralkyl imidazolecompounds such as biphenyl-methyl substituted imidazoles (e.g., EPNumber 253,310, Jan. 20, 1988); ES8891(N-morpholinoacetyl-(−1-naphthyl)-L-alanyl-(4, thiazolyl)-L-alanyl(35,45)-4-amino-3-hydroxy-5-cyclo-hexapentanoyl-N-hexylamide, SankyoCompany, Ltd., Tokyo, Japan); SKF108566(E-alpha-2-[2-butyl-1-(carboxyphenyl)methyl]1H-imidazole-5-yl[methylane]-2-thiophenepropanoic acid, Smith KlineBeecham Pharmaceuticals, PA); Losartan (DUP753/MK954, DuPont MerckPharmaceutical Company); Remikirin (RO42-5892, F. Hoffman LaRoche AG);A₂ agonists (Marion Merrill Dow) and certain non-peptide heterocycles(G.D.Searle and Company).

“Angiotensin converting enzyme,” (ACE), is an enzyme which catalyzes theconversion of angiotensin Ito angiotensin II. ACE inhibitors includeamino acids and derivatives thereof, peptides, including di- andtripeptides and antibodies to ACE which intervene in therenin-angiotensin system by inhibiting the activity of ACE therebyreducing or eliminating the formation of processor substance angiotensinII. ACE inhibitors have been used medically to treat hypertension,congestive heart failure, myocardial infarction and renal disease.Classes of compounds known to be useful as ACE inhibitors includeacylmercapto and mercaptoalkanoyl prolines such as captopril (U.S. Pat.No. 4,105,776) and zofenopril (U.S. Pat. No. 4,316,906), carboxyalkyldipeptides such as enalapril (U.S. Pat. No. 4,374,829), lisinopril (U.S.Pat. No. 4,374,829), quinapril (U.S. Pat. No. 4,344,949), ramipril (U.S.Pat. No. 4,587,258), and perindopril (U.S. Pat. No. 4,508,729),carboxyalkyl dipeptide mimics such as cilazapril (U.S. Pat. No.4,512,924) and benazapril (U.S. Pat. No. 4,410,520), phosphinylalkanoylprolines such as fosinopril (U.S. Pat. No. 4,337,201) and trandolopril.

“Renin inhibitors” are compounds which interfere with the activity ofrenin. Renin inhibitors include amino acids and derivatives thereof,peptides and derivatives thereof, and antibodies to renin. Examples ofrenin inhibitors that are the subject of United States patents are asfollows: urea derivatives of peptides (U.S. Pat. No. 5,116,835); aminoacids connected by nonpeptide bonds (U.S. Pat. No. 5,114,937); di- andtri-peptide derivatives (U.S. Pat. No. 5,106,835); amino acids andderivatives thereof (U.S. Pat. Nos. 5,104,869 and 5,095,119); diolsulfonamides and sulfinyls (U.S. Pat. No. 5,098,924); modified peptides(U.S. Pat. No. 5,095,006); peptidyl beta-aminoacyl aminodiol carbamates(U.S. Pat. No. 5,089,471); pyrolimidazolones (U.S. Pat. No. 5,075,451);fluorine and chlorine statine or statone containing peptides (U.S. Pat.No. 5,066,643); peptidyl amino diols (U.S. Pat. Nos. 5,063,208 and4,845,079); N-morpholino derivatives (U.S. Pat. No. 5,055,466);pepstatin derivatives (U.S. Pat. No. 4,980,283); N-heterocyclic alcohols(U.S. Pat. No. 4,885,292); monoclonal antibodies to renin (U.S. Pat. No.4,780,401); and a variety of other peptides and analogs thereof (U.S.Pat. Nos. 5,071,837, 5,064,965, 5,063,207, 5,036,054, 5,036,053,5,034,512, and 4,894,437).

Agents that bind to cellular adhesion molecules and inhibit the abilityof white blood cells to attach to such molecules include polypeptideagents. Such polypeptides include polyclonal and monoclonal antibodies,prepared according to conventional methodology. Such antibodies alreadyare known in the art and include anti-ICAM 1 antibodies as well as othersuch antibodies described above.

Anticoagulant agents include, but are not limited to, Ancrod;Anticoagulant Citrate Dextrose Solution; Anticoagulant Citrate PhosphateDextrose Adenine Solution; Anticoagulant Citrate Phosphate DextroseSolution; Anticoagulant Heparin Solution; Anticoagulant Sodium CitrateSolution; Ardeparin Sodium; Bivalirudin; Bromindione; Dalteparin Sodium;Desirudin; Dicumarol; Heparin Calcium; Heparin Sodium; Lyapolate Sodium;Nafamostat Mesylate; Phenprocoumon; Tinzaparin Sodium; and WarfarinSodium.

Heparin may stabilize symptoms in evolving stroke, but anticoagulantsare useless (and possibly dangerous) in acute completed stroke, and arecontraindicated in hypertensives because of the increased possibility ofhemorrhage into the brain or other organs. Although the timing iscontroversial, anticoagulants may be started to prevent recurrentcardiogenic emboli. Clot lysing agents, including tissue plasminogenactivator and streptokinase, are being evaluated for the very earlytreatment of acute stroke. Nimodipine has recently been is shown toimprove survival and clinical outcome after ischemic stroke.

Other than aspirin, ticlopidine is another antiplatelet agent that hasbeen shown to be beneficial for stroke treatment. Endarterectomy may beindicated in patients with 70 to 99 percent narrowing of a symptomaticinternal carotid artery. However, most authorities agree that carotidendarterectomy is not indicated in patients with TIAs that are referableto the basilar-vertebral system, in patients with significant deficitsfrom prior strokes, or in patients in whom a stroke is evolving.

HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase is themicrosomal enzyme that catalyzes the rate limiting reaction incholesterol biosynthesis (HMG-CoA6Mevalonate). An HMG-CoA reductaseinhibitor inhibits HMG-CoA reductase, and as a result inhibits thesynthesis of cholesterol. A number of HMG-CoA reductase inhibitors hasbeen used to treat individuals with hypercholesterolemia. More recently,HMG-CoA reductase inhibitors have been shown to be beneficial in thetreatment of stroke (Endres M, et al., Proc Natl Acad Sci USA, 1998,95:8880-5).

HMG-CoA reductase inhibitors useful for co-administration with theagents of the invention include, but are not limited to, simvastatin(U.S. Pat. No. 4,444,784); lovastatin (U.S. Pat. No. 4,231,938);pravastatin sodium (U.S. Pat. No. 4,346,227); fluvastatin (U.S. Pat. No.4,739,073); atorvastatin (U.S. Pat. No. 5,273,995); cerivastatin, andnumerous others described in U.S. Pat. Nos. 5,622,985; 5,135,935;5,356,896; 4,920,109; 5,286,895; 5,262,435; 5,260,332; 5,317,031;5,283,256; 5,256,689; 5,182,298; 5,369,125; 5,302,604; 5,166,171;5,202,327; 5,276,021; 5,196,440; 5,091,386; 5,091,378; 4,904,646;5,385,932; 5,250,435; 5,132,312; 5,130,306; 5,116,870; 5,112,857;5,102,911; 5,098,931; 5,081,136; 5,025,000; 5,021,453; 5,017,716;5,001,144; 5,001,128; 4,997,837; 4,996,234; 4,994,494; 4,992,429;4,970,231; 4,968,693; 4,963,538; 4,957,940; 4,950,675; 4,946,864;4,946,860; 4,940,800; 4,940,727; 4,939,143; 4,929,620; 4,923,861;4,906,657″ 4,906,624; and 4,897,402, the disclosures of which patentsare incorporated herein by reference.

Nitric oxide (NO) has been recognized as a messenger molecule with manyphysiologic roles, in the cardiovascular, neurologic and immune systems(Griffith, T M et al., J Am Coll Cardiol, 1988, 12:797-806). It mediatesblood vessel relaxation, neurotransmission and pathogen suppression. NOis produced from the guanidino nitrogen of L-arginine by NO Synthase(Moncada, S and Higgs, E A, Eur J Clin Invest, 1991, 21:361-374). Agentsthat upregulate endothelial cell Nitric Oxide Synthase include, but arenot limited to, L-arginine, rho GTPase function inhibitors (seeInternational Application WO 99/47153, the disclosure of which isincorporated herein by reference), and agents that disrupt actincytoskeletal organization (see International Application WO 00/03746,the disclosure of which is incorporated herein by reference).

“Co-administering,” as used herein, refers to administeringsimultaneously two or more compounds of the invention (e.g., a Fit-1,vacuolar ATPase, CD44, Lot-1, AA892598, and/or Mrg-1, nucleic acidand/or polypeptide, and an agent known to be beneficial in the treatmentof, for example, a cardiovascular condition, e.g., an anticoagulant-),as an admixture in a single composition, or sequentially, close enoughin time so that the compounds may exert an additive or even synergisticeffect, i.e., on reducing cardiomyocyte cell-death in a cardiovascularcondition.

The invention also embraces solid-phase nucleic acid molecule arrays.The array consists essentially of a set of nucleic acid molecules,expression products thereof, or fragments (of either the nucleic acid orthe polypeptide molecule) thereof, each nucleic acid molecule selectedfrom the group consisting of Fit-1, vacuolar ATPase, CD44, Lot-1,AA892598, and Mrg-1, fixed to a solid substrate. In some embodiments,the solid-phase array further comprises at least one control nucleicacid molecule. In certain embodiments, the set of nucleic acid moleculescomprises at least one, at least two, at least three, at least four, oreven at least five nucleic acid molecules, each selected from the groupconsisting of Fit-1, vacuolar ATPase, CD44, Lot-1, AA892598, and Mrg-1,provided that when only one nucleic acid molecule is present on thearray, the nucleic acid molecule is not vacuolar ATPase. In preferredembodiments, the set of nucleic acid molecules comprises a maximumnumber of 100 different nucleic acid molecules. In importantembodiments, the set of nucleic acid molecules comprises a maximumnumber of 10 different nucleic acid molecules.

According to the invention, standard hybridization techniques ofmicroarray technology are utilized to assess patterns of nucleic acidexpression and identify nucleic acid expression. Microarray technology,which is also known by other names including: DNA chip technology, genechip technology, and solid-phase nucleic acid array technology, is wellknown to those of ordinary skill in the art and is based on, but notlimited to, obtaining an array of identified nucleic acid probes (e.g.,molecules described elsewhere herein such as Fit-1, vacuolar ATPase,CD44, Lot-1, AA892598, and/or Mrg-1) on a fixed substrate, labelingtarget molecules with reporter molecules (e.g., radioactive,chemiluminescent, or fluorescent tags such as fluorescein, Cye3-dUTP, orCye5-dUTP), hybridizing target nucleic acids to the probes, andevaluating target-probe hybridization. A probe with a nucleic acidsequence that perfectly matches the target sequence will, in general,result in detection of a stronger reporter-molecule signal than willprobes with less perfect matches. Many components and techniquesutilized in nucleic acid microarray technology are presented in NatureGenetics, Vol. 21, January 1999, the entire contents of which isincorporated by reference herein.

According to the present invention, microarray substrates may includebut are not limited to glass, silica, aluminosilicates, borosilicates,metal oxides such as alumina and nickel oxide, various clays,nitrocellulose, or nylon. In all embodiments a glass substrate ispreferred. According to the invention, probes are selected from thegroup of nucleic acids including, but not limited to: DNA, genomic DNA,cDNA, and oligonucleotides; and may be natural or synthetic.Oligonucleotide probes preferably are 20 to 25-mer oligonucleotides andDNA/cDNA probes preferably are 500 to 5000 bases in length, althoughother lengths may be used. Appropriate probe length may be determined byone of ordinary skill in the art by following art-known procedures. Inone embodiment, preferred probes are sets of two or more of the nucleicacid molecules set forth as SEQ ID NOs: 1, 3, 5, 7, 9, 11 and/or 12.Probes may be purified to remove contaminants using standard methodsknown to those of ordinary skill in the art such as gel filtration orprecipitation.

In one embodiment, the microarray substrate may be coated with acompound to enhance synthesis of the probe on the substrate. Suchcompounds include, but are not limited to, oligoethylene glycols. Inanother embodiment, coupling agents or groups on the substrate can beused to covalently link the first nucleotide or olignucleotide to thesubstrate. These agents or groups may include, but are not limited toamino, hydroxy, bromo, and carboxy groups. These reactive groups arepreferably attached to the substrate through a hydrocarbyl radical suchas an alkylene or phenylene divalent radical, one valence positionoccupied by is the chain bonding and the remaining attached to thereactive groups. These hydrocarbyl groups may contain up to about tencarbon atoms, preferably up to about six carbon atoms. Alkylene radicalsare usually preferred containing two to four carbon atoms in theprincipal chain. These and additional details of the process aredisclosed, for example, in U.S. Pat. No. 4,458,066, which isincorporated by reference in its entirety.

In one embodiment, probes are synthesized directly on the substrate in apredetermined grid pattern using methods such as light-directed chemicalsynthesis, photochemical deprotection, or delivery of nucleotideprecursors to the substrate and subsequent probe production.

In another embodiment, the substrate may be coated with a compound toenhance binding of the probe to the substrate. Such compounds include,but are not limited to: polylysine, amino silanes, amino-reactivesilanes (Nature Genetics, Vol. 21, January 1999) or chromium (Gwynne andPage, 2000). In this embodiment, presynthesized probes are applied tothe substrate in a precise, predetermined volume and grid pattern,utilizing a computer-controlled robot to apply probe to the substrate ina contact-printing manner or in a non-contact manner such as ink jet orpiezo-electric delivery. Probes may be covalently linked to thesubstrate with methods that include, but are not limited to,UV-irradiation and heat.

Targets are nucleic acids selected from the group, including but notlimited to, DNA, genomic DNA, cDNA, RNA, mRNA and may be natural orsynthetic. In all embodiments, nucleic acid molecules from subjectssuspected of developing or having a cardiovascular condition, arepreferred. In certain embodiments of the invention, one or more controlnucleic acid molecules are attached to the substrate. Preferably,control nucleic acid molecules allow determination of factors includingbut not limited to: nucleic acid quality and binding characteristics;reagent quality and effectiveness; hybridization success; and analysisthresholds and success. Control nucleic acids may include, but are notlimited to, expression products of genes such as housekeeping genes orfragments thereof.

To select a set of cardiovascular disease markers, the expression datagenerated by, for example, microarray analysis of gene expression, ispreferably analyzed to determine which genes in different categories ofpatients (each category of patients being a different cardiovasculardisorder), are significantly differentially expressed. The significanceof gene expression can be determined using Permax computer software,although any standard statistical package that can discriminatesignificant differences is expression may be used. Permax performspermutation 2-sample t-tests on large arrays of data. For highdimensional vectors of observations, the Permax software computest-statistics for each attribute, and assesses significance using thepermutation distribution of the maximum and minimum overall attributes.The main use is to determine the attributes (genes) that are the mostdifferent between two groups (e.g., control healthy subject and asubject with a particular cardiovascular disorder), measuring “mostdifferent” using the value of the t-statistics, and their significancelevels.

Expression of cardiovascular disease nucleic acid molecules can also bedetermined using protein measurement methods to determine expression ofSEQ ID NOs: 2, 4, 6, 8, and/or 10, e.g., by determining the expressionof polypeptides encoded by SEQ ID NOs: 1, 3, 5, 7, and/or 9,respectively. Preferred methods of specifically and quantitativelymeasuring proteins include, but are not limited to: massspectroscopy-based methods such as surface enhanced laser desorptionionization (SELDI; e.g., Ciphergen ProteinChip System), non-massspectroscopy-based methods, and immunohistochemistry-based methods suchas 2-dimensional gel electrophoresis.

SELDI methodology may, through procedures known to those of ordinaryskill in the art, be used to vaporize microscopic amounts of tumorprotein and to create a “fingerprint” of individual proteins, therebyallowing simultaneous measurement of the abundance of many proteins in asingle sample. Preferably SELDI-based assays may be utilized tocharacterize cardiovascular conditions as well as stages of suchconditions. Such assays preferably include, but are not limited to thefollowing examples. Gene products discovered by RNA microarrays may beselectively measured by specific (antibody mediated) capture to theSELDI protein disc (e.g., selective SELDI). Gene products discovered byprotein screening (e.g., with 2-D gels), may be resolved by “totalprotein SELDI” optimized to visualize those particular markers ofinterest from among SEQ ID NOs: 1, 3, 5, 7, and/or 9. Predictive modelsof tumor classification from SELDI measurement of multiple markers fromamong SEQ ID NOs: 1, 3, 5, 7, and/or 9 may be utilized for the SELDIstrategies.

The use of any of the foregoing microarray methods to determineexpression of cardiovascular disease nucleic acids can be done withroutine methods known to those of ordinary skill in the art and theexpression determined by protein measurement methods may be correlatedto predetermined levels of a marker used as a prognostic method forselecting treatment strategies for cardiovascular disease patients.

The invention will be more fully understood by reference to thefollowing examples. These examples, however, are merely intended toillustrate the embodiments of the invention and are not to be construedto limit the scope of the invention.

EXAMPLES Example 1

Experimental Protocols: Materials and Methods

Mechanical Strain Device

Experiments of mechanically overloading cardiomyocytes have generallybeen performed by stretching cells with no control of the cardiac cycle,an approach that does not allow distinction between mechanical overloadin contraction versus relaxation. In the present study, we designed andconstructed a unique experimental system that allows preciselycontrolled mechanical strains as well as electrical pacing in culturedcardiomyocytes, to investigate, inter alia, how cardiomyocytemechanotransduction is regulated by the cardiac cycle, and identifygenes that are involved in such regulation.

The Pacing-Strain Device. The approach to mechanical stimulation used anapparatus that has multiple platens that contact the underside ofsilicone elastomer membranes to apply a spatially isotropic biaxialstrain profile to the membrane (Schaffer J L, et al., J Orthop Res,1993, 12:709-719; and U.S. Provisional Patent application filed on Jul.16, 1999 entitled “AN APPARATUS FOR STUDYING MYOCARDIAL MECHANICALOVERLOAD HYPERTROPHY AND USES THREFOR, by Richard T. Lee, and bearing.Six individual 78 mm membranes can be stretched at once with varyingamplitudes of strain by controlling displacement of each platen with astepper motor. Measured Green strains are accurate to ˜±0.25% at strainsfrom 1-14% (Cheng G C, et al., Circ Res, 1997, 80:28-36; Brown T D, JBiomechanics, 2000, 33:3-14). Throughout this study, 8% biaxial strainwas used.

To control the timing of mechanical strain relative to the cardiaccycle, the computer paced each dish electrically, and controlled: thephase between the mechanical strain and the electrical impulse, theelectrical impulse duration, and the voltage of the impulse. Inaddition, the electrical impulses had alternating polarity to minimizeelectrochemical effects such as pH gradients at the electrodes. The twooutputs were each connected to a single set of electrodes in each dish.The dishes were paced in parallel with a resistance of approximately 500ohms per dish.

The positive and negative voltage sources were provided by two powersupplies (6545A, Hewlett Packard Company, Palo Alto, Calif.). Thecontrol circuit was divided into two parts: a high voltage circuit and alow voltage or digital signal circuit. The high voltage circuit was agate that switched the output based on the input signal. The low voltagecircuit accepted two control signals from the computer and accepted thepulse width from a variable resistor, which controlled both the positiveand negative voltage gates. The low voltage circuit allowed a voltagepulse between 0-120V DC amplitude and 2-37 ms duration. Lights providedcontinuous monitoring of the pulses, and the timing of the circuits andcalibration were validated by oscilloscope.

The electrodes for each dish were two arc-shaped AgCl₂ wire electrodesat the base of the inner surface of the dish, just above the deformablemembrane. The electrodes were pre-made, ethanol-sterilized, and placedinto the dish just prior to each experiment to minimize potentialtoxicity from silver. Using this method no cellular death or detachmentwas observed in 24 hr experiments. Each arc was 120 degrees; weperformed a two dimensional finite element analysis to estimate theuniformity of the potential field with this configuration. Thesecalculations estimate a spatial variation in the potential field of{root mean square}=29%. Thus, this system provides highly uniformbiaxial mechanical strain, with a relatively small variation in thevoltage field.

Mechanical stimulation protocols. We imposed strain only during firstthird of the cardiac cycle by electrical stimulation for strain imposedduring the “systolic phase”, and only during one third of the cardiaccycle in the relaxation phase for strain imposed during “diastolicphase,” respectively. Conditions used in this study were: (1) control;(2) strain, no pacing; (3) pacing, no strain; (4) strain imposed duringsystolic phase; and (5) strain imposed during diastolic phase.

Neonatal rat ventricular myocytes (NRVM) from 1-day old Sprague-Dawleyrats were isolated by previously described methods (Springhorn J P, andClaycomb W C., Biochem J, 1989; 258:73-78; Arstall M A, et al., J MolCell Cardiol, 1998, 30:1019-25). NRVM were plated on the coated membranedish at a density of 2,000,000 cells/dish in DMEM containing 7% FCS andincubated 24 h. Approximate cell confluence was 85-90%. NRVM were thenmade quiescent by washing with 10 ml of Hanks' balanced salt solution(HBSS, 138 mM NaCl, 5.3 mM KCl, 4.0 mM NaHCO₃, 1.3 mM CaCl₂, 0.5 mMMgCl₂, 0.4 mM MgSO₄, 0.4 mM KH₂PO₄, 0.3 mM Na₂HPO₄, 5.6 mM glucose; LifeTechnologies, Inc., Rockville, Md.) twice and incubating with 26 ml ofDMEM containing 0.2% FCS for 48-72 hours.

In these cell culture conditions, cells beat at 40-60 beats/minute. Atthis rate, we have observed negligible competition when pacing at a rateof 70 beats/minute. We performed trial capture experiments; ninelocations on each dish were sampled. Capture efficiency was similar atall locations, and maximal capture occurred at 60 V and above with 10 msof pulse width. Therefore, a voltage of 70 V with 10 ms of impulseduration at a rate of 1.2 Hz (70 beats/minute) was selected. Under theseconditions we did not observe partial cell detachment.

Transcriptional Profiling. The DNA microarray experiment was performedwith rat neonatal cardiac myocytes cultured on fibronectin-coatedmembranes with serum-free medium for 48 hours. Cells were deformed withan 8% deformation imposed only during systole for a period of 30minutes, and RNA was prepared after 6 hours of subsequent no strainconditions and no pacing conditions. This time point was based uponprevious studies demonstrating that the gene tenascin (positive controlfor cardiomyocytes) is induced at this time period. The DNA microarrayhybridization experiment was performed using the Affymatrix GeneChipRGU34A (Affymetrix, Inc., Santa Clara, Calif.). Data were analyzed usingAffymatrix software.

Northern Analyses. The cDNA clones for differentially expressed geneswere obtained by PCR using the GenBank sequences. Each clone wassequenced from both 5′ and 3′ ends to confirm identity. Positiveelements in the DNA microarray were confirmed by Northern blothybridization analysis in at least three independent experiments usingthree different sources of NRVMs. Total RNA was isolated by theguanidium thiocyanate and phenol chloroform method (Chomcyznski, et al.,Anal. Biochem., 1987, 162:156-159). For Northern blotting, 15 mg RNA wasloaded on a 1.0% agarose-formaldehyde gel (2.0 mol/l), transferred to anylon membrane (Amersham Pharmacia Biotech AB, Piscataway, N.J.), and UVcross-linked with a UV Stratalinker (Stratagene, Inc., La Jolla,Calif.). Each probe was hybridized with ExpressHyb solution (ClontechLabs., Inc., Palo Alto, Calif.) at 68° C. for 1 hour. The membrane waswashed with 2×SSC, 0.05% SDS solution for 30 to 40 minutes and threetimes at room temperature and 0.1×SSC, 0.1% SDS solution with continuousshaking at 50° C. for 40 minutes. The membrane was exposed to film at−80° C., and radiographs were scanned and analyzed with Optimas 5.0software (Optimas Co./Media Cybernetics, Silver Springs, Md.).Densitometric units were normalized to the ethidium-stained 28Sribosomal subunit on the membrane.

Results. FIG. 1 shows the timecourne (early, left; late, right) of theinduction of Fit-1 mRNA expression by 8% cyclic mechanical strain inneonatal cardiac myocytes in culture. Maximal induction occurs at 3hours and is sustained for 15 hours.

FIG. 2 shows the effects of 8% mechanical strain, angiotensin receptorblockade (ARB, CP-19116, 100 nM), angiotensin II (Ang II, 50 nM),interleukin-1β (IL-1β, 10 ng/ml), and phorbal ester (Pma, 200 nM) for 3hours on the induction of Fit-1 mRNA expression in cultured neonatal ratcardiac myocytes. The induction of Fit-1 mRNA expression by strain wasnot blocked by angiotensin receptor blockade; furthermore, treatmentwith angiotensin II did not induce Fit-1 mRNA expression. Treatment withboth IL-1β and PMA were associated with an induction of Fit-1 mRNAexpression in the absence of mechanical strain.

FIG. 3 shows the effects of 8% mechanical strain, hydrogen peroxide(H₂O₂, 100 uM) and the antioxidant, TIRON (10 mN) on the iduction ofFit-1 mRNA expression. Unlike the mRNA expression of the mechanicallyinduced Tenascin-C gene which is induced by H₂O₂ in the absence ofmechanical strain and blocked by TIRON, H₂O₂ does not induce Fit-1 inthe absence of strain and blocks the strain-induced induction of Fit-1.TIRON slightly attenuated the mRNA expression of Fit-1 in the absenceand presence of strain.

FIG. 4 shows the effects of actinomycin D (5 μg/ml, left) andcyclohexamide (10 μg/ml, right) on the induction of Fit-1 mRNAexpression by 8% mechanical strain. Actinomycin D and cyclohexamide wereapplied during mechanical strain. Actinomycin D blocked the induction ofFit-1 mRNA expression at both 2 and 4 hours suggesting that theinduction of Fit-1 in response to strain is due to increasedtranscription of Fit-1. The protein synthesis inhibitor, cyclohexamideblocked the induction of Fit-1 mRNA expression in response to strainsuggesting that new protein synthesis is required for the induction ofFit-1 mRNA expression.

FIG. 5 shows the effects of 8% mechanical strain alone and incombination with interleukin-1β (IL-1β, 10 ng/ml), and phorbal ester inthe absence of strain (PMA, 100 ng/ml) on Fit-1 mRNA expression incultured neonatal cardiac myocytes. Both IL-1β and mechanical strainalone induced Fit-1 mRNA expression but the induction of Fit-1 bymechanical strain in the presence of IL-1β was not further increasedsuggesting that mechanical strain and IL-1β do not act in a synergisticor additive manner on the induction of Fit-1. The strongest induction ofFit-1 mRNA expression is seen with PMA. The rank order potency for theinduction of Fit-1 mRNA expression is PMA>strain>IL-1β.

FIG. 6 shows neonatal rat cardiac myocytes were exposed to 8% strain for0, 1, 3, 6, 9, hours. Total RNA was isolated using RNeasy kit. Five μgof total RNA were size-separated on 1% agarose-formaldehyde gel andtransferred to nylon membrane. After cross-linking with UV light,membrane was hybridized with ³²P-labeled probe specific for V-ATPase Bsubunit. The membrane was then exposed to x-ray film for 3 hours at −80°C. with an intensifying screen.

Example 2

Introduction

Cytokines and Cardiac Injury. Stress-activated cytokines participate inmany forms of cardiac injury and pathophysiological conditions, the mostcharacterized ones being tumor necrosis factor-α, interleukin-1 andinterleukin-6. These molecules are not constitutively expressed in thenormal heart but are rapidly induced during ischemia and reperfusion orupon hemodynamic overloading, suggesting that they play an importantrole in the initial myocardial response to stress, injury or growthstimuli (Mann D L, Cytokine and Growth Factor Reviews. 1996; 7:341-354;St. John Sutton M G, et al. Circulation. 2000; 101:2981-2988). However,cytokines have also been shown to be stably expressed in pathologicmyocardial conditions including ischemic heart disease and heart failureand are associated with a poor prognosis (Pulkki K J, et al. Annals ofMedicine. 1997; 29:339-343; Kubota T, et al Proc Natl Acad. Sci. 1998;95:6930-6935; Aukrust P, et al. Am J Cardiol 1999; 83:376-382; MacGowanG A, et al. Am J Cardiol 1997; 79:1128-1132; Roig E, et al. Am J Cardiol1998; 688-690; Tsutamoto T, et al. J Am Coll Cardiol 1998; 31:391-398;Prabhu S D, et al. Circulation. 2000; 101:2103-2109; Murray D R, et al.Annu Rev Immunol. 2000; 18:451-494).

Interleukin-1 signaling through the interleukin-1 receptor is an earlyevent in inflammatory cytokine signaling in many different systems(Trehu E G., Clin Cancer Res. 1996; 8:1341-51). In cardiac injury,interleukin-6 is produced by cardiac myocytes secondary to stimulationwith interleukin-1, tumor necrosis factor-α, or lipopolysaccharide andhas been detected in the post-ischemic lymph during reperfusion ofischemic myocardium (Gwechenberger M, et al. Circulation 1999;99:546-551). Recently recognized is the potential expression ofcounteracting anti-inflammatory cytokines in cardiac disease secondaryto interleukin-1 signaling. Interleukin-4 and interleukin-10 cansuppress the synthesis of tumor necrosis factor-α and enhance therelease of soluble tumor necrosis factor receptors, which are ligandsinks for tumor necrosis factor (Joyce D A., 1994; Eur. J. Immunol.11:2699-705). Interleukin-10 is increased in patients with heart failure(Yamaoka M, et al. Jpn Circ J. 1999; 63:951-956) and interleukin-10serum levels are increased when tumor necrosis factor-α serum levels areincreased in patients with dilated cardiomyopathy (Ohtsuka T, et al. JAm Coll Cardiol. 2001; 37:412-417).

T1/ST2 (fit-1): A Novel Mechanically Induced Receptor. We haveidentified a novel potential stress-activated signaling pathway in theheart: regulation of the induction of an interleukin-1 family membergene, T1/ST2. Little is known of the induction, signaling and functionof T1/ST2 in any cell type and T1/ST2 was shown in separate areas ofinvestigation to have two seemingly unrelated functions. One of these isgrowth regulation and the other is immune modulation. Both compensatoryhypertrophic growth and immune/inflammatory modulation are involved inthe pathophysiology of cardiovascular diseases.

Growth. The T1/ST2 gene was first identified by its induction followingserum stimulation of resting mouse 3T3 fibroblasts, suggesting that theT1/ST2 gene participates in growth regulation (Tominaga S., FEBS Letters1989; 258:301-304). The same group later identified a longer transcriptconsisting of transmembrane and cytoplasmic domains homologous to thefull-length interleukin-1 receptor (Yanagisawa K, et al. FEBS Letters.1993; 318:83-87).

Immunity. T1/ST2 is expressed on T helper-2, but not T helper-1, cellsof the adaptive immune system, which produce interleukin-4,interleukin-5 and interleukin-10 (Yanagisawa K I, et al. J Biochem.1997; 121:95-103; Coyle A J, et al. J Exp Med. 1999; 190:895-902). Thelper-2 cells mediate beneficial responses to infection, but aredetrimental in the development of allergy and asthma. There is a strongcorrelation between expression of T1/ST2 and interleukin-4 production onT helper-2 cells (Coyle A J, et al. J Exp Med. 1999; 190:895-902).T1/ST2 plays a critical role in differentiation to and activation of Thelper-2 but not T helper-1 cells (O'Neill L A J, et al. ImmunologyToday. 2000; 21:206-209).

Inhibition of T1/ST2 signaling attenuated T helper 2-mediated inductionof eosinophil inflammatory responses in lung and inhibited cytokinesecretion from T helper-2 cells without modifying interferon-gammasecretion from T helper-1 cells (Coyle A J, et al. J Exp Med. 1999;190:895-902). These studies indicate that expression of T1/ST2 can alterthe cytokine profile in favor of expression of interleukin-4,interleukin-5 and interleukin-10. Interleukin-10 has recently been shownto have anti-inflammatory effects in the setting of cardiac injury(Ohtsuka T, et al. J Am Coll Cardiol. 2001; 37:412-417). Similarly, theabsence of T1/ST2 expression could result in a shift towardsinterferon-gamma expression, which may be deleterious followingmyocardial injury.

Taken together, the involvement of T1/ST2 in growth responses and immunefunction coupled with the clinical recognition of the role of cytokinesin the inflammatory response to ischemia/reperfusion are suggestive thatT1/ST2 activation is a growth- or stress-activated signaling pathwaythat contributes to myocardial growth and remodeling.

Phenotype of T1/ST2 Null Mice. (Townsend M J, et al. J Exp Med. 2000;191: 1069-1075). The absence of T1/ST2 in T1/ST2 null mice does notcompromise their basal immune function in the absence of immunechallenge. However, T1/ST2 null mice have an impaired ability togenerate IL-4, IL-5, and IL-10, but not IFN-γ (a Th1 cytokine) and togenerate a T helper-2 inflammatory response during eosinophilicinfiltration in the lung (a Th2 response).

We have begun to study the induction of T1/ST2 in cardiac myocytes andits involvement in survival/death signaling within the context of themyocyte signaling pathways. Preliminary studies presented below showthat T1/ST2 is induced in cardiac myocytes in response to interleukin-1and mechanical strain and that the induction of T1/ST2 by interleukin-1may be dependent on NF-κB activation. T1/ST2 mRNA is also induced inhuman adult vascular smooth muscle cells in response to interleukin-1.T1/ST2 protein is expressed in the mouse heart early after myocardialischemia in vivo as well as in human aorta tissue from patients withunstable plaque.

Results:

IN VITRO STUDIES. The following studies demonstrate the induction ofT1/ST2 by mechanical strain and interleukin-1, possibly throughactivation of NF-κB. Both transcripts of T1/ST2 (that is, Fit-1S andFit-1M) are induced by strain in cardiac myocytes. T1/ST2 mRNA isinduced by mechanical strain in cultured neonatal cardiac myocytes (FIG.8).

T1/ST2 mRNA is induced by mechanical strain in cultured neonatal cardiacmyocytes. Neonatal rat ventricular myocytes were isolated by collagenasedigestion, plated on fibronectin-coated silicone membrane dishes at adensity of 3.5 million cells/dish in 13 ml media as previously described(Yamamoto K, et al. J Biol. Chem. 1999; 274:21840-21846). This techniqueyields cultures with ≧95% myocytes. Mechanical deformation was appliedusing a device that provides uniform biaxial cyclic strain as previouslydescribed (Yamamoto K, et al. J Biol. Chem. 1999; 274:21840-21846). RNAwas extracted (Qiagen) and Northern blotting was performed using as aprobe a ³²P-labelled 600 bp PCR fragment specific to rat T1/ST2. Maximalinduction occurs at 3 hours, is sustained for 9 hours and declines by 15hours.

Both interleukin-1β and mechanical strain each induce T1/ST2 RNA incardiac myocytes (FIG. 9). Shown is the induction of T1/ST2 byinterleukin-1 and strain. We also found that the induction of T1/ST2 bymechanical strain in the presence of interleukin-1β was not furtherincreased suggesting that interleukin-1 does not sensitize myocytes tothe effects of mechanical strain (or vice versa) on the induction ofT1/ST2. The 1 hour time point was included in the event that inductionby strain is saturated at 3 hours and therefore masks an additive effectof interleukin-1β. Shown in the two right lanes are the effects ofphorbol ester (PMA) at 1 and 3 hours. The rank order potency for theinduction of T1/ST2 mRNA expression is PMA>strain>interleukin-1β. Sinceinterleukin-1β signals through NF-κB and PMA through PKC these resultssuggest that NF-κB and PKC activation both participate in the inductionof T1/ST2.

T1/ST2 may be a NF-κB target gene in cardiac myocytes throughinterleukin-1/interleukin-1 receptor signaling (FIG. 10). Previouslyreported by us (Yamamoto K, et al. J Biol. Chem. 1999; 274:21840-21846),mechanical strain of cardiac myocytes activates NF-κB. To investigatethe role of NF-κB in interleukin-1β and strain induction of T1/ST2 RNA,we overexpressed IκBα, which decreases NF-κB DNA binding activity.Cultured cardiac myocytes were infected with IκBα overexpressionadenovirus vector or with β-galactosidase control vector and exposed for4 hours to 8% cyclic mechanical strain or interleukin-1 (10 ng/ml). RNAwas analyzed by Northern blotting with ³²P-labeled Fit-1 cDNA probe.Ectopic expression of IκBα blocked interleukin-1β induction of T1/ST2-1mRNA and partially blocked strain induction of T1/ST2 mRNA expressionwhen compared with T1/ST2 induction in cells treated with theβ-galactosidase control vector. These results suggest that T1/ST2 is anearly, NF-κB target gene through interleukin-1/interleukin-1 receptorsignaling. In contrast, pathways in addition to NF-κB activation may beinvolved in the induction of T1/ST2 RNA by mechanical strain. T1/ST2mRNA is also induced by interleukin-1 but not PMA or tumor necrosisfactor (TNF) in human adult vascular smooth muscle cells.

In addition to the above-noted results, we have shown that T1/ST2 isinduced secondary to NF-κB activation by interleukin-1 and NF-κB islinked to cardiac myocyte survival. Further in vitro studies areperformed to confirm that T1/ST2 activation is linked to cell growth andsurvival.

IN VIVO STUDIES. In vivo Expression of T1/ST2 Protein in MyocardialInfarction in Mice. FIG. 11 shows protein expression of T1/ST2 usingimmunohistochemistry in paraffin sections of mouse hearts 1 and 3 dayspost-infarction. T1/ST2 protein was visualized by DAB staining. Thisantibody (Morwell Diagnostics) does not distinguish between the twoisoforms of T1/ST2. Positive staining (brownish color) is seen 1 daypost-infarction (post-MI) in the normal, infarct and border zones butnot at 3 days post-MI. These results suggest that ST2 protein is rapidlyexpressed in response to myocardial injury during the early phase ofpost-infarction remodeling before the migration of macrophages into theinfarct and border zones (see 3 days post-MI). Magnification: 40×.

In addition to the above, we are generating an operational colony ofT1/ST2 null mice. Our in vivo studies indicate that T1/ST2 is expressedin the mouse heart following myocardial infarction. The in vivo studiesconfirm the hypothesis that local cardiac expression of T1/ST2 favorablymodifies the process of LV remodeling following ischemia and leftventricular pressure overload. We have also generated adeno-associatedviruses to express isoforms of these genes and their effects on nullmice are determined.

More recently, we have obtained results which support the utility of thegene/protein called fit-1, or ST-2, as a diagnostic indicator of acardiovascular condition in humans. We assayed serum levels on 69patients who participated in the HEART study, a clinical trial of acutemyocardial infarction patients. The assay employed a monoclonal assayfor the human ST2 protein. The results show that the levels of ST2correlated with serum creatine phosphokinase levels, which is a standardway of looking at size of heart attack. Also, such levels rapidlydecline after the infarct. The levels were: Day 1: 3.8+/−3.3 ng/ml; Day14: 0.9+/−0.5; and Day 90: 0.8+/−0.5 and are highly statisticallysignificant. These results also establish that the protein is secretedduring heart attacks and can be easily measured, thereby supporting theasserted utility of the invention.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references disclosed herein are incorporated by reference in theirentirety.

What is claimed is presented below and is followed by a SequenceListing.

I claim:
 1. A method for treating a subject to reduce the risk ofdeveloping a cardiovascular condition, the method comprising:determining a level of secreted ST2 in a biological sample from asubject at risk of developing a cardiovascular condition; comparing thelevel of secreted ST2 in the biological sample to a control; identifyingthe subject having an elevated level of secreted ST2 relative to acontrol; and administering to the identified subject an agent thatreduces the risk of the cardiovascular condition.
 2. The method of claim1, wherein the cardiovascular condition is characterized by mechanicalstrain, mechanical overload, or mechanically-induced deformation incardiac cells or tissue.
 3. The method of claim 1, wherein thecardiovascular condition is selected from the group consisting of:cardiac hypertrophy, myocardial infarction, stroke, arteriosclerosis,and heart failure.
 4. The method of claim 1, wherein the biologicalsample is blood or serum.
 5. The method of claim 1, wherein thedetermining is performed using an antibody or an antibody fragment thatbinds to secreted ST2.
 6. The method of claim 5, wherein the antibody orantibody fragment is labeled with a radioactive label or an enzyme. 7.The method of claim 1, wherein the subject is a human.
 8. The method ofclaim 1, wherein the control is a predetermined level.
 9. The method ofclaim 1, wherein the control is a level of secreted ST2 in a healthysubject.
 10. The method of claim 1, wherein the subject ishypercholesterolemic, hypertriglyceridemic, hyperlidemic, a smoker, orhypertensive.
 11. The method of claim 1, wherein the agent is selectedfrom the group consisting of: an anti-inflammatory agent, ananti-thrombotic agent, an anti-platelet agent, a fibrinolytic agent, alipid-reducing agent, a direct thrombin inhibitor, a glycoproteinIIb/IIIa receptor inhibitor, a calcium channel blocker, abeta-adrenergic receptor blocker, a cyclooxygenase-2 inhibitor, andrenin-angiotensin system (RAS) inhibitors.
 12. The method of claim 11,wherein the renin-angiotensin system inhibitor is selected from thegroup consisting of: an angiotensin-converting enzyme (ACE) inhibitor,an angiotensin II receptor blocker (ARB), an angiotensin II receptorantagonist, an agent that activates the catabolism of angiotensin II,and an agent that prevents the synthesis of angiotensin I.
 13. Themethod of claim 1, wherein the agent is administered orally, rectally,topically, nasally, intradermally, transdermally, by infusion,intravenously, or intramuscularly.
 14. The method of claim 1, whereinthe agent is formulated for timed-release, delayed-release, orsustained-release.